Polymerase-template complexes

ABSTRACT

The present disclosure provides methods and compositions for enhancing the processivity of a polymerase in catalyzing template-dependent DNA synthesis in high concentrations of salt. Also disclosed are methods and compositions for enhancing the assembly of polymerase-template complex compatible with active DNA synthesis in the presence of low levels of nucleotides and at a high temperature, such as temperatures at or near the melting temperature of the polymerase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication No. 62/406,431 filed Oct. 11, 2016 and U.S. ProvisionalPatent Application No. 62/301,607 filed Feb. 29, 2016, the disclosuresof which are each incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 1, 2017, isnamed 04338_531US1_SL.TXT and is 60,572 bytes in size.

TECHNICAL FIELD

The present disclosure relates generally to methods and compositions forimproving DNA sequencing processivity, and more particularly toenhancing sequencing yield in a DNA sequencing reaction via adjustmentof temperature, nucleotide concentration, and/or polymeraseconcentration.

BACKGROUND

Nanopores have recently emerged as a label-free platform forinterrogating sequence and structure in nucleic acids. Data aretypically reported as a time series of ionic current as DNA sequence isdetermined when an applied electric field is applied across a singlepore controlled by a voltage-clamped amplifier. Hundreds to thousands ofmolecules can be examined at high bandwidth and spatial resolution.

A crucial obstacle to the success of nanopores as a reliable DNAanalysis tool is the processivity or average read length. Efficientbinding of template by the polymerase, for example, is key to highsequencing yield. This and other desirable properties can be enhanced bymodifying polymerases to increase the amount of sequence informationobtained from a template-dependent sequencing reaction. Additionally,processivity can also be increased by providing conditions that favorthe formation (or stabilize) the polymerase-template complexes. Due tothe numerous varying conditions at which the sequencing reaction can beran, however, the specific variables (conditions) that optimize certainsequencing reactions, such as nanopore based sequencing, have remainedlargely elusive.

SUMMARY OF THE INVENTION

Provided herein are methods and compositions that can be used tooptimize sequencing reactions, such as nanopore-based sequencing. Incertain example aspects, provided are methods and compositions thatutilize high salt concentration to enhance a sequencing reaction. In oneaspect, for example, provided is a method is provided for preparing apolymerase-template complex. The method comprises (a) providing apolymerase; and (b) contacting the polymerase with a polynucleotidetemplate in a solution comprising a high concentration of salt and beingessentially free of nucleotides, thereby preparing thepolymerase-template complex.

In another aspect, a method is provided for increasing the processivityof a template-polymerase complex, the method comprising forming atemplate-polymerase complex in a solution comprising a highconcentration of salt and being essentially free of nucleotides; whereinthe processivity of the template-polymerase complex is greater than theprocessivity of the same template-polynucleotide complex when formed ina solution comprising an equally high concentration of salt and in thepresence of nucleotides. For example, the processivity is increased by afaster rate of association of the template with the polymerase, and/orthe processivity is increased by a slower rate of dissociation of thetemplate from the polymerase.

In another aspect, a method is provided for performingtemplate-dependent DNA synthesis, the method comprising: (a) providing apolymerase-template complex in a solution comprising a highconcentration of salt and being essentially free of nucleotides; and (b)initiating template-dependent DNA synthesis by adding nucleotides to thesolution.

In another aspect, a method is provided for nanopore sequencing at highsalt concentration, the method comprising: (a) providing apolymerase-template complex in a solution comprising a highconcentration of salt, the solution being essentially free ofnucleotides; (b) combining the polymerase-template complex with ananopore to form a nanopore-sequencing complex; (c) providing taggednucleotides to the nanopore sequencing complex to initiatetemplate-dependent nanopore sequencing of the template at highconcentration of salt; and (d) detecting with the aid of the nanopore, atag associated with each of the tagged nucleotides during incorporationof each of the nucleotides while each of the nucleotides is associatedwith the polymerase, thereby determining the sequence of thepolynucleotide template.

In each of the various foregoing aspects, the polymerase may be avariant polymerase, such as a polymerase comprising an amino acidsequence having at least 70% sequence identity to the polymerase of SEQID NO:2. Further, the nanopore can be a monomeric nanopore, such as anOmpG nanopore, or the nanopore can be an oligomeric nanopore such as analpha-hemolysin nanopore. Moreover, the high concentration of salt isdefined, for example, as a salt concentration of at least 100 mM.

In another aspect, a storage or reaction composition is provided, thestorage or reaction composition comprising a polymerase-template complexin a solution of at least 100 mM salt. In some embodiments, thecomposition is essentially free of nucleotides.

In certain other example aspects, provided are methods and compositionsthat utilize low nucleotide concentrations and high temperatures toenhance a sequencing reaction. For example, in ore aspect provided is amethod for preparing a polymerase-template complex. The method includes,for example, providing a polymerase and then contacting the polymerasewith a polynucleotide template in a solution, thereby preparing thepolymerase-template complex. The solution includes a low concentrationof nucleotides and has a high temperature.

In another aspect, provided is a method for increasing processivity of atemplate-polymerase complex. The method includes forming apolymerase-template complex in a solution—the solution including a lowconcentration of nucleotides and having a high temperature. In suchmethods, the processivity of the polymerase-template complex formed inthe high-temperature solution is greater than a processivity resultingfrom a control polymerase-template complex solution at room temperature.

In another aspect, provided is a method for nanopore-based sequencing ofa polynucleotide template. The method includes forming apolymerase-template complex in a solution—the solution including a lowconcentration of nucleotides having a high temperature. The formedpolymerase-template complex is combined with a nanopore to form ananopore-sequencing complex. Tagged nucleotides are provided to thenanopore sequencing complex to initiate template-dependent nanoporesequencing of the template at the high temperature. With the aid of thenanopore, a tag associated with each of the tagged nucleotides isdetected during incorporation of each of the tagged nucleotides whileeach of the tagged nucleotides is associated with the polymerase,thereby determining the sequence of the polynucleotide template. Thenanopore may be a monomeric nanopore, such as an OmpG nanopore, or amultimeric nanopore, such as an alpha-hemolysin based nanopore.

In each of the foregoing aspects involving low nucleotide concentrationsand high temperatures, the method may further include saturating thesolution with the polymerase of the polymerase-template complex. Thepolymerase, for example, may be a polymerase variant, such as apolymerase having 85%, 90%, 95%, 98% or more sequence identity to theamino acid sequence set forth as SEQ ID NO: 2. In certain aspects, thelow concentration of the nucleotides is between is 0.8 μM to 2.2 μM,such as 1.2 μM. In certain aspects, the high temperature is above roomtemperature, such as 35° C. to 45° C. In certain aspects, the hightemperature is 40° C.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing in color. Copiesof this patent or patent publication with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 is an illustration of the minimal catalytic steps required forsingle-nucleotide incorporation by DNA polymerase. The reaction beginswith the binding of free DNA polymerase enzyme (E) to a duplexprimer/template DNA complex (DNA_(n)) resulting in a binary enzyme-DNAcomplex (E•DNA_(n)). k_(on,DNA) denotes the rate of association of theenzyme with the template; and k_(off,DNA) denotes the rate ofdissociation of the enzyme from the enzyme-DNA complex. The equilibriumdetermined by the k_(on,DNA) and k_(off,DNA) rates defines the staticprocessivity of the polymerase-template complex. Thus, the staticprocessivity of the enzyme can be increased by an increase in the rateof association, k_(on,DNA), and/or a decrease in the rate ofdissociation, k_(off,DNA) Association of the correct nucleotide (dNTP)in the presence of divalent cations, such as Mg²⁺, promotes theenzyme-DNA-dNTP ternary complex formation (E•DNA_(n)•dNTP•Mg²⁺). Thek_(on, nucleotide) denotes the rate of nucleotide binding of the enzyme.The k_(off, nucleotide) denotes the rate of nucleotide dissociation formthe enzyme template complex. The equilibrium determined by thek_(on, DNA) and k_(off, DNA) while the polymerase is extending thetemplate defines the replicative processivity of the polymerase. Thus,the replicative processivity of the polymerase can be increased by anincrease in the rate of DNA association, k_(on, DNA), and/or a decreasein the rate of DNA dissociation, k_(off, DNA). The binding of the dNTPinduces the first conformational change of the enzyme in the ternarycomplex. A phosphodiester bond is formed between the a-phosphate of theincoming dNTP and the 3′-OH of the template/primer terminus to producean added nucleotide base to the primer terminus (E*•DNA_(n+1)•PP_(i)).The reaction generates a pyrophosphate (PP_(i)) and a proton. A secondconformational change allows for the release of the PP_(i) to complete acycle of nucleotide incorporation.

FIG. 2 is an illustration showing an exemplary template used in a FRETdisplacement assay.

FIG. 3 is a graph showing exemplary results of the effect of formingpolymerase-template complex in the presence of polyphosphate nucleotideson the rate of association of template with polymerase at variousconcentrations of salt. Reference is made to Example 3.

FIG. 4A-4C is a series of graphs showing association curves for thefluorescence signals shown in FIG. 3. Reference is made to Example 3.

FIG. 5A-5B is a series of graphs showing results of the effect ofblocked nucleotides on inhibiting the formation of polymerase-templatecomplex. Fluorescence signal obtained in a FRET assay are shown in (A),and dissociation curves are shown in (B). Reference is made to Example4.1.

FIG. 6A-6B is a series of graphs showing exemplary results of the effectof nucleotides on the formation of polymerase-template complex and rateof dissociation of template from polymerase-template complex formed inthe presence Mg2+(♦) or 20 uM d6Ps (polyphosphate nucleotides; ▪).Fluorescence signal obtained in a FRET assay are shown in (A), anddissociation curves are shown in (B). Reference is made to Example 4.2.

FIG. 7A-7B is a series of graphs showing exemplary results of the effectof nucleotides (d6Ps) on the formation of polymerase-template complexand rate of dissociation of template from polymerase-template complexformed in the absence (♦) or presence (▪) of Ca2+. Fluorescence signalobtained in a FRET assay are shown in (A), and dissociation curves areshown in (B). Reference is made to Example 4.3.

FIG. 8A-8B is a series of graphs showing exemplary results of the rateof dissociation of template from polymerase-template complex whencomplex was formed in the presence of Mg2+(▪), 20 uM polyphosphatenucleotides (Δ), or in the absence of both Mg2+ and 20 uM polyphosphatenucleotides (♦). Fluorescence signal obtained in a FRET assay are shownin (A), and dissociation curves are shown in (B). Reference is made toExample 4.4.

FIG. 9A-9B is a series of graphs showing exemplary results of the rateof dissociation of template from polymerase-template complex whencomplex was formed in the presence of dNTPs (▪), or d6Ps (♦).Fluorescence signal obtained in a FRET assay are shown in (A), anddissociation curves are shown in (B). Reference is made to Example 4.5.

FIG. 10A is an image of a Native 5% TBE gel showing static binding ofpolymerase to template at room temperature. The polymerase concentrationis increased (0, 1×, 4×, and 8×) relative to template concentration, inthe absence of nucleotides. At 4× and 8× polymerase concentrations, theband shifts indicate non-specific binding of multiple polymerases tomultiple locations on the template. Reference is made to Example 5.

FIG. 10B is an image of a Native 5% TBE gel showing static binding ofpolymerase to template at 40° C. Like FIG. 10A, the polymeraseconcentration is increased (0, 1×, 4×, and 8×) relative to templateconcentration, but in the presence of 1.2 μM nucleotides(polyphosphate). The lack of band shifts at 4× and 8× concentrationsindicates specific binding of the polymerase to the 3′ end of thetemplate DNA at 40° C. Reference is made to Example 5.

FIGS. 11A-11C illustrate the correlation between polymerase-templatebinding and extension of the template at 40° C. More particularly, FIG.11A is an image of a Native 5% TBE gel showing binding of polymeraseconcentration at 0, 1×, 2×, 4×, 6×, and 8× to template. As shown,increasing polymerase results in an increase in template binding at 40°C. and in the presence of 1.2 μM nucteotides. FIG. 11B is an image of aNative 5% TBE gel showing extension of the template, following bindingshown in FIG. 11A. As evidenced by the shifts in band intensity from thelower band to the upper band with increased concentration of polymerase,increasing the concentration of polymerase results in increased templateextension (the extension occurring in the presence of 10 μMnucleotides). FIG. 11C is a graph showing the correlation of templatebinding (from FIG. 11A) with template extension (FIG. 11B). As shown,the % bound correlates directly with the % extension (slope=1).Reference is made to Example 6.

FIGS. 12A-12D are a series of graphs illustrating template extensionfollowing the formation of the polymerase-template complex at 40° C. andin the presence of low levels of nucleotides (1.2 NM). FIG. 12A showsamplitude curves for the fluorescence signal obtained in a FRET assay,with increasing concentration of polymerase (0, 1×, 2×, 4×, 6×, 8×, and1×) to template. As shown, increasing polymerase concentration atbinding results in increased extension (as evidenced the by increasedsignal amplitude at increased concentrations compared to controls). FIG.12B shows the amplitude quantification of the fluorescent signals forthe data in FIG. 12A, i.e., the fluorophore-quencher (extensionreaction) (▪) as compared to the fluorophore alone (▴). FIG. 12C showsthe percent extension (♦) of the polymerase at increasing polymeraseconcentrations, as determined by comparing the fluorescent amplitude ofthe fluorophore-quencher (extension reaction) to the fluorescentamplitude of the fluorophore alone. FIG. 12D shows template extensioncomparison between the gel based assay (see above) and the plate reader(FRET) assay. As shown, there is good correlation between % templateextension as measured by gel-based or plate-reader based assays. ForFIGS. 12A-12D, Reference is made to Example 7.

FIGS. 13A-13B are a series of graphs illustrating polymerase templatebinding and dissociation at varying binding conditions at 40° C. FIG.13A shows the amplitude curves for the fluorescence signal obtained in aFRET assay for the binding conditions indicated. FIG. 13B shows thedissociation curves for 1.2 μM dNpCpp (blocking nucleotides)/3 mM Sr⁺²(♦); 1.2 μM dNpCpp alone (▪); 1.2 μM polyphosphate nucleotides (⋄)alone; or no nucleotides/Sr (x). As shown, Sr⁺² does not impactpolymerase-template dissociation. A low concentration of polyphosphatenucleotides (⋄) provides the lowest level of dissociation. Reference ismade to Example 8.

FIGS. 14A-14B are graphs illustrating the effects of salt concentrationon polymerase-complex formation at 40° C. and dissociation at 30° C. inthe presence and absence of high nucleotide concentration (36 uM). FIG.14A shows the dissociation curve obtained from a FRET assay at 75 mMKGlu in the presence of nucleotides (▪) (final concentration 10 μM) andin the absence of nucleotides (control) (♦). FIG. 14B shows thedissociation curve obtained from a FRET assay at 380 mM KGlu in thepresence of nucleotides (▪) (final concentration 10 μM) and in theabsence of nucleotides (control) (♦). As shown, the amount of templatebound at time zero is roughly 2-fold better in the absence ofnucleotides. Hence, at both salt concentrations, the presence of highnucleotide concentration (10 uM+during binding) decreasespolymerase-template binding. Reference is made to Example 9.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Practitioners areparticularly directed to Sambrook et al., 1989, and Ausubel F M et al.,1993, for definitions and terms of the art. It is to be understood thatthis invention is not limited to the particular methodology, protocols,and reagents described, as these may vary.

Numeric ranges are inclusive of the numbers defining the range. The termabout is used herein to mean plus or minus ten percent (10%) of a value.For example, “about 100” refers to any number between 90 and 110.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Definitions

The term “processivity” herein refers to the ability of a polymerase toremain attached to the template and perform multiple modificationreactions. “Modification reactions” include but are not limited topolymerization, and exonucleolytic cleavage. In some embodiments,“processivity” refers to the ability of a DNA polymerase to perform asequence of polymerization steps without intervening dissociation of theenzyme from the growing DNA chains. Typically, “processivity” of a DNApolymerase is measured by the number of nucleotides (for example 20 nts,300 nts, 0.5-1 kb, or more) that are incorporated i.e. polymerized by apolymerase into a growing DNA strand prior to the dissociation of theDNA polymerase from the growing DNA strand. The processivity of DNAsynthesis by a DNA polymerase is defined as the number of nucleotidesthat a polymerase can incorporate into DNA during a single template□binding event, before dissociating from a DNA template. The overallefficiency of DNA synthesis increases when the processivity of apolymerase increases. “Processivity” can depend on the nature of thepolymerase, the sequence of a DNA template, and reaction conditions, forexample, salt concentration, temperature or the presence of specificproteins. As used herein, the term “high processivity” refers to aprocessivity higher than 20 nts (e.g., higher than 40 nts, 60 nts, 80nts, 100 nts, 120 nts, 140 nts, 160 nts, 180 nts, 200 nts, 220 nts, 240nts, 260 nts, 280 nts, 300 nts, 320 nts, 340 nts, 360 nts, 380 nts, 400nts, or higher) per association/dissociation with the template. Thehigher the processivity of a polymerase, that greater the number ofnucleotides that can be incorporated prior to dissociation of thepolymerase from the template, and therefore, the longer the sequence(read length) that can be obtained. Processivity can be measuredaccording the methods defined herein and in WO 01/92501 A1 (MJ Bioworks,Inc., Improved Nucleic Acid Modifying Enzymes, published 6 Dec. 2001).Processivity encompasses static processivity and replicativeprocessivity.

The term “static processivity” herein refers to the permanence of apolymerase-template complex in the absence of nucleotide incorporationi.e. in the absence of polynucleotide synthesis, as determined by therate of association of polymerase with template, k_(on,DNA), and therate of dissociation of polymerase from the polymerase-template complexk_(off,DNA). Static processivity is defined in the absence ofpolynucleotide synthesis.

The term “replicative processivity” herein refers to the permanence of apolymerase-template complex in the during nucleotide incorporation i.e.in the presence of polynucleotide synthesis, as determined by the rateof association of polymerase with template, k_(on, nucleotide), and therate of dissociation of polymerase from the polymerase-template complexk_(off, nucleotide).

As used herein, the term “association rate,” when used in reference to agiven polymerase, herein refers to the rate at which a polymeraseassociates with a template. The association rate can be interpreted as atime constant for association (“k_(on, DNA)”) of a polymerase with anucleic acid template under a defined set of reaction conditions. Someexemplary assays for measuring the dissociation time constant of apolymerase are described further below In some embodiments, thedissociation time constant can be measured in units of inverse time,e.g., ^(sec−1) or min⁻¹.

The term “dissociation rate,” when used in reference to a givenpolymerase, herein refers to the rate at which a polymerase dissociatesfrom the template of the polymerase-template complex. The dissociationrate can be interpreted as a time constant for dissociation(“k_(off, DNA)”) of a polymerase from a nucleic acid template under adefined set of reaction conditions. Some exemplary assays for measuringthe dissociation time constant of a polymerase are described furtherbelow. In some embodiments, the dissociation time constant can bemeasured in units of inverse time, e.g., ^(sec−1) or min⁻¹.

The term “stability” when used in reference to a polymerase-templatecomplex, herein refers to the permanence of a polymerase-templatecomplex, as determined by the rates of association and dissociation ofthe template to and from the polymerase.

The term “read length” herein refers to the number of nucleotides that apolymerase incorporates into a nucleic acid strand in atemplate-dependent manner prior to dissociation from the template.

The term “high concentration of salt” herein refers to a concentrationof salt, i.e., monovalent salt that is at least 100 mM and up to 1 Msalt.

The term “salt-tolerant” is used herein in reference to a polymeraseenzyme that retains polymerase activity in a solution comprising a highsalt concentration e.g. greater than 100 mM salt.

The term “essentially free of nucleotides” herein refers to a solutionthat is at least 99.9% free of nucleotides.

The terms “polynucleotide” and “nucleic acid” are herein usedinterchangeably to refer to a polymer molecule composed of nucleotidemonomers covalently bonded in a chain. Single stranded DNA (ssdeoxyribonucleic acid; ssDNA), double stranded DNA (dsDNA) and RNA(ribonucleic acid) are examples of polynucleotides.

The term “amino acid” in its broadest sense, herein refers to anycompound and/or substance that can be incorporated into a polypeptidechain. In some embodiments, an amino acid has the general structureH₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally-occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a D-aminoacid; in some embodiments, an amino acid is an L-amino acid. “Standardamino acid” refers to any of the twenty standard L-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, and/or substitution with otherchemical without adversely affecting their activity. Amino acids mayparticipate in a disulfide bond. The term “amino acid” is usedinterchangeably with “amino acid residue.” and may refer to a free aminoacid and/or to an amino acid residue of a peptide. It will be apparentfrom the context in which the term is used whether it refers to a freeamino acid or a residue of a peptide. It should be noted that all aminoacid residue sequences are represented herein by formulae whose left andright orientation is in the conventional direction of amino-terminus tocarboxy-terminus.

The term “nanopore sequencing complex” or “nanopore complex” hereinrefers to a nanopore linked to an enzyme, e.g., a polymerase, which inturn is associated with a polymer, e.g., a polynucleotide or a protein.The nanopore sequencing complex is positioned in a membrane, e.g., alipid bilayer, where it functions to identify polymer components, e.g.,nucleotides or amino acids.

The term “polymerase-template complex” herein refers to a polymerasethat is associated/coupled with a polymer, e.g., polynucleotidetemplate.

The term “complexed polymerase” herein refers to a polymerase that isassociated with a polynucleotide template in a polymerase-templatecomplex.

The term “nucleotide” herein refers to a monomeric unit of DNA or RNAconsisting of a sugar moiety (pentose), a phosphate, and a nitrogenousheterocyclic base. The base is linked to the sugar moiety via theglycosidic carbon (1′ carbon of the pentose) and that combination ofbase and sugar is a nucleoside. When the nucleoside contains a phosphategroup bonded to the 3′ or 5′ position of the pentose it is referred toas a nucleotide. A sequence of operatively linked nucleotides istypically referred to herein as a “base sequence” or “nucleotidesequence,” and is represented herein by a formula whose left to rightorientation is in the conventional direction of 5′-terminus to3′-terminus.

The term “nucleotide analog” herein refers to analogs of nucleosidetriphosphates, e.g., (S)-Glycerol nucleoside triphosphates (gNTPs) ofthe common nucleobases: adenine, cytosine, guanine, uracil, andthymidine (Horhota at al. Organic Letters, 8:5345-5347 [2006]).

The term “tag” herein refers to a detectable moiety that may be atoms ormolecules, or a collection of atoms or molecules. A tag may provide anoptical, electrochemical, magnetic, or electrostatic (e.g., inductive,capacitive) signature, which may be detected with the aid of a nanopore.

The term “tagged nucleotide” herein refers to a nucleotide having a tagattached at its terminal phosphate.

The term “blocked nucleotide” herein refers to a modifiednon-incorporable nucleotide that blocks primer extension. dNpCpp is anexample of a “blocked nucleotide.”

The term “polymerase” herein refers to an enzyme that catalyzes thepolymerization of nucleotide (i.e., the polymerase activity). The termpolymerase encompasses DNA polymerases, RNA polymerases, and reversetranscriptases. A “DNA polymerase” catalyzes the polymerization ofdeoxynucleotides. An “RNA polymerase” catalyzes the polymerization ofribonucleotides. A “reverse transcriptase” catalyzes the polymerizationof deoxynucleotides that are complementary to an RNA template. As usedherein, the term “polymerase” and its variants comprise any enzyme thatcan catalyze the polymerization of nucleotides (including analogsthereof) into a nucleic acid strand. Typically but not necessarily suchnucleotide polymerization can occur in a template-dependent fashion.

The terms “template DNA molecule” and “template strand” are usedinterchangeably herein to refer to a strand of a nucleic acid from whicha complementary nucleic acid strand is synthesized by a DNA polymerase,for example, in a primer extension reaction.

The term “sample polynucleotide” herein refers to a polynucleotideobtained from a sample, e.g., a biological sample.

The term “template-dependent synthesis” refers to a process thatinvolves the synthesis of a new DNA strand (e.g., DNA synthesis by DNApolymerase) that is complementary to a template strand of interest. Theterm “template-dependent synthesis” typically refers to polynucleotidesynthesis of RNA or DNA wherein the sequence of the newly synthesizedstrand of polynucleotide is dictated by complementary base pairing (see,for example, Watson, J. D. et al., In: Molecular Biology of the Gene,4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987)).

The term “nanopore” herein refers to a channel or passage formed orotherwise provided in a membrane. A membrane may be an organic membrane,such as a lipid bilayer, or a synthetic membrane, such as a membraneformed of a polymeric material. The nanopore may be disposed adjacent orin proximity to a sensing circuit or an electrode coupled to a sensingcircuit, such as, for example, a complementary metal oxide semiconductor(CMOS) or field effect transistor (FET) circuit. In some examples, ananopore has a characteristic width or diameter on the order of 0.1 Nmto about 1000 nm. Some nanopores are proteins. OmpG and alpha-hemolysinare examples of a protein nanopore.

The terms “alpha-hemolysin,” “α-hemolysin,” “aHL,” “αHL,” “a-HL” and“α-HL” are used interchangeably and herein refer to a protein thatself-assembles into a heptameric water-filled transmembrane nanoporechannel.

The term “OmpG” herein refers to an Outer Membrane Protein G monomericnanopore.

The term “nanopore sequencing” herein refers to a method that determinesthe sequence of a polynucleotide with the aid of a nanopore. In someembodiments, the sequence of the polynucleotide is determined in atemplate-dependent manner.

The term “monomeric nanopore” herein refers to a nanopore protein thatconsists of a single subunit, OmpG is an example of a monomericnanopore.

The term “oligomeric nanopore” herein refers to nanopores that can becomposed of multiple identical subunits, multiple distinct subunits, ora mixture of identical and distinct subunits. Nanopores with identicalsubunits are termed “homo-oligomeric nanopores”. Nanopores containingtwo or more distinct polypeptide subunits are termed “hetero-oligomericnanopores”. Alpha-hemolysin is an example of an oligomeric nanopore.

The term “wild-type” herein refers to a gene or gene product (e.g., aprotein) that has the characteristics of that gene or gene product whenisolated from a naturally occurring source.

The term “parental” or “parent” herein refers to a protein, e.g., ananopore or enzyme, to which modifications, e.g., substitution(s),insertion(s), deletion(s), and/or truncation(s), are made to producevariants thereof. This term also refers to the polypeptide with which avariant is compared and aligned. The parent may be a naturally occurring(wild type) polypeptide, or it may be a variant thereof, prepared by anysuitable means.

The term “mutation” herein refers to a change introduced into a parentalsequence, including, but not limited to, substitutions, insertions,deletions (including truncations). The consequences of a mutationinclude, but are not limited to, the creation of a new character,property, function, phenotype or trait not found in the parentalsequence.

The term “variant” herein refers to a modified protein e.g. a variantPol6 polymerase, which displays altered characteristics when compared tothe parental protein, e.g., altered processivity.

The term “purified” herein refers to a polypeptide that is present in asample at a concentration of at least 95% by weight, or at least 98% byweight of the sample in which it is contained.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used.

For ease of reference, polymerase variants of the application aredescribed by use of the following nomenclature: Original amino acid(s):position(s): substituted amino acid(s). According to this nomenclature,for instance the substitution of serine by an alanine in position 242 isshown as:

-   -   Glu585Lys or E585K.

Multiple mutations are separated by plus signs, i.e.:

-   -   Glu585Lys+Leu731Lys or E585K+L731K        representing mutations in positions 585 and 731 substituting        glutamic acid and Leucine acid for Lysine and Leucine for        Lysine, respectively.

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as: E585K/R or E585K or E585R.

Example Embodiments

In certain example embodiments, the present disclosure provides methodsand compositions for enhancing the processivity of the polymerase duringtemplate-dependent polynucleotide synthesis in the presence of a highconcentration of salt. In other example embodiments, the presentdisclosure provides methods and compositions for enhancing theprocessivity of the polymerase during template-dependent polynucleotidesynthesis in the presence of low nucleotide concentrations and hightemperatures. The methods and compositions provided are applicable tomethods of template-dependent DNA synthesis, including DNA amplificationand sequencing. Sequencing methods include sequencing-by-synthesis ofsingle polynucleotide molecules, such as nanopore sequencing of singleDNA molecules.

As illustrated in FIG. 1, the processivity of a polymerase, such as aDNA polymerase, is directly related to the formation of thepolymerase-template complex and the incorporation of dNTP by the enzyme.Under these parameters, the overall processivity of the polymerase isdependent on the static and replicative processivity. The greater thestatic and/or the replicative processivity of the polymerase, thegreater the overall processivity of the polymerase. As shown in FIG. 1,the static processivity is determined by the rate of association(k_(on, DNA)) and dissociation (k_(off, DNA)) of the polymerase with thetemplate. Static processivity is determined in the absence ofpolynucleotide synthesis. Thus, the greater (or faster) the rate ofassociation of polymerase with template, and/or the lesser (or slower)the rate of dissociation of polymerase from template, the greater thestatic processivity of the polymerase.

Replicative processivity is determined in the presence of nucleotidesand based on the rate of association and dissociation of nucleotide withthe polymerase of the polymerase-template complex. Thus, the greater (orfaster) the rate of association of nucleotide with the complexedpolymerase, and/or the lesser (or slower) the rate of dissociation ofnucleotide from the complexed polymerase, the greater the replicativeprocessivity of the polymerase. The replicative processivity isdetermined by the rate of association (k_(on, nucleotide)) and rate ofdissociation (k_(off, nucleotide)) of nucleotide from thepolymerase-template complex under conditions of polymerization, such asin the presence of nucleotides and divalent cation such as Mg²⁺. Thestatic processivity of a polymerase can be increased by an increase inthe association of polymerase with template to form thepolymerase-template complex, and/or a decrease in the dissociation ofpolymerase from the polymerase-template complex.

In exemplary assays, as described in the Examples herein, theassociation and dissociation rate of a given polymerase with and from atemplate can be measured by incubating the polymerase with a labeledoligonucleotide including a fluorescent label (FIG. 2) under definedconditions. When the oligonucleotide is not bound by polymerase, thefluorescence of the fluorescent label on the oligonucleotide isquenched; binding of the polymerase to the oligonucleotide results inde-quenching of the oligonucleotide label and a resulting increase influorescence. Blocking is initiated by adding an unlabeled competitoroligonucleotide to the reaction mixture; as polymerase dissociates fromthe fluorescently labeled oligonucleotide, the competitoroligonucleotide hybridizes to oligonucleotide and prevents furtherbinding of the polymerase. Fluorescence of the reaction mixture ismeasured at various time points following addition of the competitoroligonucleotide. The observed fluorescence (in RFU or relativefluorescence units) is graphed (Y axis) against time (X axis). Tocompare association and dissociation rates of a polymerase underdifferent conditions, the enzyme can be employed in a parallel andseparate reactions in which the fluorescence of each reaction mixture ismeasured at various time points, following which the dissociation ratesfor each enzyme can be calculated using any suitable method, andcompared.

Published methods describe that binding of template to polymerase toform the polymerase-template complex is carried out in the presence ofnucleotides as nucleotides have been utilized to stabilize thepolymerase-template complex. For example, US20150167072 provides methodsfor the purification of polymerase-template complexes, which includenucleotides and nucleotide analogs in the purification process tostabilize the polymerase-template complex. Similarly, US20150368626provides methods for performing nucleic acid sequencing that includescontacting a polymerase with a nucleic acid template in the presence ofone or more nucleotides.

Surprisingly, Applicant has determined that, at high concentrations ofsalt, nucleotides affect the formation of the polymerase-templatecomplex by interfering with the binding of template to the polymerase(Example 4). Additionally, Applicant has determined that binding oftemplate to polymerase in the presence of nucleotides (at other thanvery low concentrations) increase the dissociation rate of template fromthe polymerase (Example 5 and 10). The effect of nucleotides on thestatic processivity of the polymerase-template complex is not alleviatedby divalent cations such as Ca2+, which is typically included as astabilizer of polymerase-template complexes.

The destabilizing effect of high levels of nucleotides on the staticprocessivity of the polymerase-template complex is notable fortemplate-dependent synthesis of polynucleotides under conditions thatrequire synthesis to occur at high concentration of salt e.g. nanoporesequencing. In nanopore sequencing, high salt concentrations boost thesignal to noise ratio for ionic-current-based nanopore measurements.However, the high salt concentrations destabilize the polymerase-DNAtemplate complex, resulting in high polymerase turnover rates anddiminished detection of sequential nucleotide additions i.e.processivity or length of sequence reads, during polymerizationreactions is diminished.

Thus, in some embodiments, a method is provided for preparing apolymerase-template complex that comprises providing a polymerase, andcontacting the polymerase with a polynucleotide template in a solutioncomprising a high concentration of salt and being essentially free ofnucleotides. The polymerase of the polymerase-template complex can be awild-type or a variant polymerase that retains polymerase activity athigh concentration of salt. Examples of polymerases that find use in thecompositions and methods described herein include the salt-tolerantpolymerases described elsewhere herein. In some embodiments, thepolymerase of the polymerase-template complex is a Pol6 polymerase thathas an amino acid sequence that is at least 70% identical to SEQ IDNO:2.

While higher levels of nucleotides adversely affect polymerase-templatebinding, the Applicant has also surprisingly found that low levels ofnucleotides at the time of binding, along with initiating the binding athigh temperature, results in improved polymerase-template binding andresultant processivity. For example, the melting temperature of Pol6 isapproximately 40° C., and with template bound, the melting temperatureis approximately 43° C. By binding polymerase to template at 40° C., themethods and compositions provided herein promote specific binding ofpolymerase to 3′ end and denaturation of polymerase that is unbound orthat is bound to non-specific sites on the template. Applicant has alsodetermined that the improved binding is associated with improvedextension of the template (Examples 6-10).

Thus, in certain example embodiments, a method is provided for preparinga polymerase-template complex in the presence of low levels ofnucleotides and at a high temperature. Additionally, the reactionsolution can be saturated with polymerase. The polymerase of thepolymerase-template complex can be a wild-type or a variant polymerasethat retains polymerase activity at low nucleotide concentrations and athigh temperature. In certain example embodiments, the polymerase mayalso be salt resistant. In some embodiments, the polymerase of thepolymerase-template complex is a Pol6 polymerase that has an amino acidsequence that is at least 70% identical to SEQ ID NO:2. Polymerases thatare useful in the methods and compositions described herein, as well asother features, uses, and aspects of the invention are described below.

Polymerases of Polymerase-Template Complexes

In certain example embodiments, the polymerase of thepolymerase-template complexes described herein can be a DNA polymeraseand may include bacterial DNA polymerases, eukaryotic DNA polymerases,archaeal DNA polymerases, viral DNA polymerases, and phage DNApolymerases.

In certain example embodiments, the polymerase of thepolymerase-template complex can be a naturally occurring polymerase andany subunit and truncation thereof, mutant polymerase, variantpolymerase, recombinant, fusion or otherwise engineered polymerase,chemically modified polymerase, synthetic molecule, and any analogs,homologs, derivatives or fragments thereof that retain the ability toperform template-dependent polynucleotide synthesis. Optionally, thepolymerase can be a mutant polymerase comprising one or more mutationsinvolving the replacement of one or more amino acids with other aminoacids, the insertion or deletion of one or more amino acids from thepolymerase, or the linkage of parts of two or more polymerases.

In some embodiments, the polymerase used to prepare thepolymerase-template complex is a salt-tolerant polymerase capable ofcatalyzing template-dependent DNA synthesis in a solution comprising ahigh salt concentration and being essentially free of nucleotides. Thehigh salt concentration at which the polymerase-template complex can beformed is defined as a salt concentration of at least 100 mM salt e.g.100 mM potassium glutamate (K-glu).

Salt-tolerant polymerases can be wild-type or variants of polymerasesthat are naturally salt-tolerant. In some embodiments, salt-tolerantpolymerases are type B DNA polymerases that include members of theextreme halophiles, and variants thereof as described, for example, inUS Patent Publication US2014/0113291, entitled “Salt-tolerant DNApolymerases,” which is incorporated herein by reference in its entirety.

In other embodiments, salt-tolerant polymerases can be polymerases thatare not naturally salt-tolerant, but that have been modified to becomesalt-tolerant.

In certain example embodiments, and in addition to a slow k_(off, DNA)fast k_(on, DNA). the polymerases of the polymerase-template complex cancarry out DNA polymerization at high concentrations of salt, and canhave one or more desired characteristic that find use in sequencing DNA,such as slow k_(off, nucleotide), fast k_(on, nucleotide), highfidelity, low exonuclease activity, DNA strand displacement, k_(chem),increased stability, increased processivity, salt tolerance, andcompatibility with attachment to nanopore. In certain exampleembodiments, the polymerases have the ability to incorporate apolyphosphates having 4, 5, 6, 7 or 8 phosphates, such asquadraphosphate, pentaphosphate, polyphosphate, heptaphosphate oroctophosphate nucleotide, sequencing accuracy, and long read lengths,i.e., long continuous reads.

In certain example embodiments, the polymerase may be a polymerase thatfunctions at temperatures above room temperature, such as a polymerasethat functions above about 30° C. In other example embodiments, thepolymerase may function at temperatures of 40° C. or above. Suchpolymerase may include any of the polymerases described herein thatfunction at such temperatures.

In certain example embodiments, the polymerase of thepolymerase-template complex is a polymerase that has been engineered tohave increase processivity. Such example polymerases may further includeadditional modifications that impart or enhance one or more of thedesired characteristics of a polymerase for sequencing polynucleotides(e.g. DNA).

In certain example embodiments, the engineered polymerase can be avariant Pol6 polymerase that displays increased processivity whencompared to the parental Pol6 from which it is derived. For example, theparental polypeptide is a wild-type Pol6 polypeptide. The variant Pol6polypeptide of the polymerase-template complex can be derived from awild-type parental Clostridium phage phiCPV4 wild type sequence (SEQ IDNO:1) nucleic acid coding region plus a His-tag; SEQ ID NO:1, proteincoding region) and available elsewhere (National Center forBioinformatics or GenBank Accession Numbers AFH27113). A wild-typeparental Pol6 polymerase can be a homolog of the parent Pol6 fromClostridium that can be used as a starting point for providing variantpolymerases having increased processivity.

As those skilled in the art will appreciate, other polymerases having ahigh degree of homology to the Clostridium phage sp. strain phiCPV4 mayserve as a parental Pol6 without defeating the scope of the compositionsand methods provided herein. Homologs of the parental Pol6 fromClostridium phage can share sequence identity with the Pol6 fromClostridium phage (SEQ ID NO:1) of at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99%. Forexample, a variant Pol6 can be derived from a homolog of the Clostridiumphage that is at least 70% identical to the parental Pol6 fromClostridium phage.

In other example embodiments, the variant Pol6 polymerase of thepolymerase-template complex is a variant Pol6 polypeptide that can bederived from a variant parental Pol6. In some example embodiments, thevariant parental Pol6 polymerase is the Pol6 polymerase of SEQ ID NO:2.In other embodiments, the variant parental Pol6 polymerase comprisesmodifications that remove/decrease the exonuclease activity of thepolymerase (e.g., U.S. Provisional Patent Application 62/301,475, titled“Exonuclease Deficient Polymerases,” filed on Feb. 29, 2016, which isexpressly incorporated herein by reference). In yet other embodiments,the polymerase can be mutated to reduce the rate at which the polymeraseincorporates a nucleotide into a nucleic acid strand (e.g., a growingnucleic acid strand). In some cases, the rate at which a nucleotide isincorporated into a nucleic acid strand can be reduced byfunctionalizing the nucleotide and/or template strand to provide sterichindrance, such as, for example, through methylation of the templatenucleic acid strand. In some instances, the rate is reduced byincorporating methylated nucleotides. In other embodiments, the parentalpolypeptide is a Pol6 variant to which additional mutations have beenintroduced to improve the desired characteristics of a polymerase usedin nanopore sequencing. In certain example embodiments, the variant Pol6can share sequence identity with the parental Pol6 of SEQ ID NO:2 of atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99%.

In certain example embodiments, the modification of one or more aminoacids at the DNA binding site can be one or more of a substitution, adeletion or an insertion, which modification(s) retain the polymeraseactivity of the variant polymerase, and decrease the rate ofdissociation of polynucleotide from the Pol-DNA complex relative to thatof the parent Pol6. The amino acid modification(s) can be made at one ormore of amino acid residues corresponding to amino acid residues V173,N175, N176, N177, I178, V179, Y180, S211, Y212, I214, Y338, T339, G340,G341, T343, H344, A345, D417, I418, F419, K420, I421, G422, G434, A436,Y441, G559, T560, Q662, N563, E566, E565, D568, L569, I570, M571, D572,N574, G575, L576, L577, T578, F579, T580, G581, S582, V583, T584, Y596,E587, G588, E590, F591, V667, L668, G669, Q670, L685, C687, C688, G689,L690, P691, S692, A694, L708, G709, Q717, R718, V721, I734, I737, M738,F739, D693, L731, F732, T733, T287, G288, M289, R290, T291, A292, S293,S294, I295, Y342, V436, S437, G438, Q439, E440, E585, T529M, S366A,A547F, N545L, Y225L, and D657R of SEQ ID NO:2.

In some example embodiments, the variant Pol6 enzyme having polymeraseactivity, comprises an amino acid sequence at least 70% identical tothat of the full-length parental Pol6 of SEQ ID NO:2, and has amodification at one or more of amino acids corresponding to amino acidresidues V173, N175, N176, N177, 1178, V179, Y180, S211, Y212, I214,Y338, T339, G340, G341, T343, H344, A345, D417, I418, F419, K420, I421,G422, G434, A436, Y441, G559, T560, Q662, N563, E566, E565, D568, L569,I570, M571, D572, N574, G575, L576, L577, T578, F579, T580, G581, 8582,V583, T584, Y596, E587, G588, E590, F591, V667, L668, G669, 0670, L685,C687, C688, G689, L690, P691, 8692, A694, L708, G709, Q717, R718, V721,I734, I737, M738, F739, D693, L731, F732, T733, T287, G288, M289, R290,T291, A292, S293, S294, 1295, Y342, V436, S437, G438, Q439, E440, E585,T529M, S366A, A547F, N545L, Y225L, and D657R of SEQ ID NO:2.

In some example embodiments, the mutation of one or more amino acids ofthe DNA binding site is a substitution to a positively charged aminoacid. For example, any one or more of amino acids corresponding to aminoacid residues V173, N175, N176, N177, I178, V179, Y180, S211, Y212,I214, Y338, T339, G340, G341, T343, H344, A345, D417, I418, F419, K420,I421, G422, G434, A436, Y441, G559, T560, Q662, N563, E566, E565, D568,L569, I570, M571, D572, N574, G575, L576, L577, T578, F579, T580, G581,S582, V583, T584, Y596, E587, G588, E590, F591, V667, L668, G669, Q670,L685, C687, C688, G689, L690, P691, S692, A694, L708, G709, Q717, R718,V721, I734, I737, M738, F739, D693, L731, F732, T733, T287, G288, M289,R290, T291, A292, S293, S294, I295, Y342, V436, S437, G438, Q439, E440,and E585 of SEQ ID NO:2 can be mutated to a K, R, H, Y, F, W, and/or T.

In some example embodiments, the mutation of the one or more amino acidsof the DNA binding site is a substitution to K. For example, the variantPol6 polymerase can comprise amino one or more of amino acidsubstitutions G438K, E565K, E585K, L731K, and M738K. In some exampleembodiments the variant Pol6 polymerase comprises substitution E585K. Inother example embodiments, the Pol6 polymerase comprises substitutionsE585K+L731K. In yet other embodiments, the Pol6 polymerase comprisessubstitutions E585K+M738K. In other embodiments, at least two, at leastthree, at least four, at least five, at least six amino acids or more ofthe DNA binding site are mutated.

In certain example embodiments, the mutation of the one or more aminoacids of the DNA binding site is a substitution including one or more ofT529M, S366A, A547F, N545L, Y225L, or D657R. For example, the variantpolymerase may include the following substitutions: T529M, S366A, A547F,N545L, Y225L, and D657R. In certain example embodiments, the variantpolymerase is an amino acid sequence that is about 70%, 80%, 90%, 95%,98% or more identical to the amino acid sequence set forth as SEQ ID NO:14, while retaining one or more of the substitutions identified in SEQID NO: 14 (such as retaining all the substitutions identified therein).

In certain example embodiments, the resulting variant Pol6 enzymesretain polymerase activity, and display a decreased rate of dissociationof polynucleotide form the Pol-DNA complex relative to the rate ofdissociation displayed in the parent polymerase that lacks the samemutations. In some example embodiments, the modification of the parentPol6 produces a variant Pol6 polymerase having a rate of dissociationfrom the template that is at least 2-fold less that of the parent Pol6.Modifications of the parent Pol6 can produce variant Pol6 polymeraseshaving a rate of dissociation from the template that is at least 3-foldless that of the parent Pol6, at least 4-fold less that of the parentPol6, at least 5-fold less that of the parent Pol6, at least 6-fold lessthat of the parent Pol6, at least 7-fold less that of the parent Pol6,at least 8-fold less that of the parent Pol6, at least 9-fold less thatof the parent Pol6, at least 10-fold less that of the parent Pol6.

DNA sequences encoding a wild-type parent Pol6 may be isolated from anycell or microorganism producing the Pol6 in question, using variousmethods well known in the art. Examples of DNA sequences that encodewild-type Clostridium phage phiCPV4 (i.e. wild-type Pol6), are providedherein as nucleotides 28-2220 of SEQ ID NO:3, and as nucleotides 421 to2610 of SEQ ID NO:5. In addition to the wild-type Pol6, SEQ ID NO:3comprises at its 5′ end nucleotides that encode a histidine tag (His₆;HHHHHH; SEQ ID NO:9). SEQ ID NO:5 comprises at its 5′ end nucleotidesthat encode histidine tag (His₆ (SEQ ID NO: 9)) and a SpyCatcher peptideSGDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO:10). In certain example embodiments, any ofthe polymerase identified herein, including any of the variantpolymerases, may be linked directly or indirectly to the SpyCatcherpeptide (SEQ ID NO:10).

The DNA sequence may be of genomic origin, mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genomic and cDNAorigin, prepared by ligating fragments of synthetic, genomic or cDNAorigin (as appropriate, the fragments corresponding to various parts ofthe entire DNA sequence), in accordance with standard techniques. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al. (1988).

Formation of Polymerase-Template Complex at High Salt Concentration

In certain example embodiments, the polymerase-template complex can beformed in the presence of high concentration of salt of at least 100 mMand up to 1 M salt e.g. KCl, K-glu or other monovalent salt. The highconcentration of salt can be about 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800 mM, 900 mM or greater. Typical salts include salts of metalelements. The high salt solutions can include one or more of a potassiumsalt, sodium salt, cesium salt, calcium salt, cobalt, nickel, aluminum,manganese, zinc, and lithium. Salts can also include the bicarbonate,sulfate, chloride, carbonate, nitrate, nitrite, bromide, citrate,acetate, cyanide, oxide or phosphate salt of a metal element known tothose of skill in the art. In some embodiments, the salt is potassiumglutamate (K-glu), potassium chloride (KCl), potassium sulfate (K₂SO₄),potassium nitrate (KNO₃), cesium chloride (CsCl), or cesium nitrate(CsNO₃). In some embodiments, the high salt solution includes K-Glu(potassium glutamate) or other monovalent salt. In addition, a saltuseful in the invention can include a mixture or blend of salts. Blendsof mineral salts that can be used in the invention include K-Glu andKCl, K-Glu and K₂SO₄, K-Glu and KNO₃, K-Glu and CsCl, K-Glu and CsNOs,K-Glu and KNO₃, K-Glu and CsCl, K-Glu and CsNO₃, K-Glu and CsCl, K-Gluand CsNO₃, KCl and K₂SO₄, KCl and KNO₃, KCl and CsCl, KCl and CsNO₃,K₂SO₄ and KNO₃, K₂SO₄ and CsCl, K2SO₄ and CsNO₃, KNO₃ and CsCl, KNO₃ andCsNOs, and CsCl and CsNO₃. The foregoing salts may be used in thesequencing polymerization reactions at a concentration in the range of50 to 1M, in the range of 100 to 800 mM, in the range of 200 to 700 mM,in the range of 300 to 600 mM, in the range of 400 to 500 mM. In someembodiments, the high salt concentration can be of at least 150 mM andup to 500 mM. In some embodiments, the high concentration of salt is atleast 500 mM salt.

The rate of polymerization of the complexed polymerase e.g. variant Pol6polymerase, at high salt concentrations is at least 1 base/second, atleast 5 bases/second, at least 10 bases/second, at least 20bases/second, at least 30 bases/second, at least 40 bases/second, atleast 50 bases/second, or more. In some embodiments, the rate ofpolymerization of the complexed polymerase e.g. variant Pol6 polymeraseis at least 1 base/second at 100 mM salt, 1 base/second at 200 mM salt,at least 1 base/second at 300 mM salt, at least 1 base/second at 400 mMsalt, at least 1 base/second at 500 mM salt, at least 1 base/second at600 mM salt, at least 1 base/second at 700 mM s alt, at least 1base/second at 800 mM salt, at least 1 base/second at 800 mM salt, atleast 1 base/second at 900 mM salt, at least 1 base/second at 1M salt.In some embodiments, the rate of polymerization of the complexedpolymerase e.g. variant Pol6 polymerase is between 1 and 10 bases/secondat 100 mM salt, between 1 and 10 bases/second at 200 mM salt, between 1and 10 bases/second at 300 mM salt, between 1 and 10 bases/second at 400mM salt, between 1 and 10 bases/second at 500 mM salt, between 1 and 10bases 600 mM salt, between 1 and 10 bases at 700 mM salt, between 1 and10 bases/second at 800 mM salt, between 1 and 10 bases/second at 800 mMsalt, between 1 and 10 bases/second at 900 mM salt, or between 1 and 10bases/second at 1M salt.

In some embodiments, the solution for preparing a polymerase-templatecomplex comprising a high concentration of salt further comprises apolymerase-template complex stabilizer. Examples of polymerase-templatecomplex stabilizers include without limitation Ca²⁺. Thus, in someembodiments, the solution that is provided for preparing apolymerase-template complex comprises, for example, a high concentrationof salt of between 100 mM and 500 mM K-glu. Solutions for preparingpolymerase-template complexes are essentially free of nucleotides.

Formation of a polymerase-template complex can be assayed according tovarious methods known in the art. For example, formation of thepolymerase-template complex can be determined according to the methoddescribed in Example 3.

Thus, in some embodiments, a method is provided for preparing apolymerase-template complex that comprises providing a polymerase, andcontacting the polymerase with a polynucleotide template in a solutioncomprising a high concentration of salt and being essentially free ofnucleotides. The polymerase of the polymerase-template complex can be awild-type or a variant polymerase that retains polymerase activity athigh concentration of salt. Examples of polymerases that find use in thecompositions and methods described herein include the salt-tolerantpolymerases described elsewhere herein. In some embodiments, thepolymerase of the polymerase-template complex is a Pol6 polymerase thathas an amino acid sequence that is at least 70% identical to SEQ IDNO:2.

Formation of Polymerase-Template Complexes at High Temperature and LowNucleotide Concentration

In certain example embodiments, the polymerase-template complex can beformed in the presence of high temperature, along with lowconcentrations of nucleotides. For example, in certain exampleembodiments provided is a method for preparing a polymerase-templatecomplex, the method including (a) providing a polymerase and (b)contacting the polymerase with a polynucleotide template in a solutionthat includes a low concentration of nucleotides and that is at a hightemperature, thereby preparing the polymerase-template complex.

With regard to temperature, for example, the temperature of the solutionfor forming the polymerase-template complex can be above roomtemperature, i.e, above about 20° C. For example, the high temperaturemay be about 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37°C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46°C., 47° C., 48° C., 49° C., 50° C., or more. In certain exampleembodiments, the high temperature is 38° C., 39° C., 40° C., 41° C., or42° C. In certain example embodiments, the high temperature is at ornear the melting temperature of the polymerase or thepolymerase-template complex.

While in certain example embodiments described herein the reactionsolution in which the polymerase-template complex is formed includeshigh salt and is essentially free of nucleotides, in other exampleembodiments the solution includes a low concentration of nucleotides.For example, the low nucleotide concentration may range from 0.5 μM to2.5 μM. In other example embodiments, the nucleotide concentration is0.8 μM to 2.2 μM, such as about 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM,1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM,or 2.2 μM. In addition to the low concentration of nucleotides, thesolution can include the high temperature as described herein. As anexample, the reaction solution in which the polymerase-template isformed may include the template, nucleotides at a concentration of about0.8 μM to 2.2 μM, with the solution being about 38° C. to 42° C.

To facilitate binding of the polymerase to the template, the polymerasemay, in certain example embodiments, be equal to the templateconcentration or be in molar excess of the template concentration. Forexample, the polymerase may be 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×or more of the template concentration. In other words, in certainexample embodiments, the reaction solution can be saturated withpolymerase.

Examples of polymerases of the polymerase-complex that find use in thecompositions and methods described herein include the variouspolymerases described herein. These include, for example, any of thehigh-temperature-suitable polymerases described herein, as well as thevariant polymerases described herein. In certain example embodiments,such as those described in the Examples 10-14, the polymerase includesthe sequence set forth as SEQ ID NO: 14. In other example embodiments,the polymerase of the complex has at least 70% or more identity to theamino acid sequence set forth as SEQ ID NO: 2. In certain exampleembodiments, the polymerase, or variant polymerase, can be linkeddirectly or indirectly to the SpyCatcher peptide (SEQ ID NO:10) to forma fusion peptide. As an example, the sequence set forth as SEQ ID NO:14, or a sequence having 70% or more identity thereto, may be joined,directly or indirectly, to the sequence set forth as SEQ ID NO: 10. Thepolymerase and SpyCatcher peptide may be joined, for example, by anylinker peptide known in the art.

To form the polymerase complex, for example, the polymerase, template,and nucleotides are brought in contact with each other in a reactionsolution at the desired temperature. The complexes are then allowed toform in the solution. For example, the reaction solution may beincubated for about 10, 15, 20, 25, 30 minutes or more before sequencingis initiated. Once the polymerase-template complexes are formed andsequencing is initiated, for example, additional nucleotides may beadded to the solution, thereby raising the concentration of thenucleotides in the solution. That is, once sequencing is initiated, itis not necessary to maintain the low concentration of nucleotides toachieve the several benefits described herein, e.g., increasedpolymerase-template complex formation and enhanced processivity. Forexample, the concentration of the nucleotides may be raised to about 5μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, or 15 μM.In certain example embodiments, the reaction solution may also include ahigh salt solution as described herein.

Like the evaluation of polymerase-template complex formation withincreased salt, the formation of the polymerase-template complex can beassayed according to various methods known in the art. For example,formation of the polymerase-template complex can be determined accordingto the method described in Example 3 (i.e., using a FRET assay). Usingthe methods and compositions described herein, for example, formation ofthe polymerase-template complex may be increased by about 10%, 15%, 20%,20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared to a control thatlacks low nucleotides and/or that is ran at or below room temperature.

In certain example embodiments, provided herein is a method forincreasing processivity of a template-polymerase complex, the methodcomprising forming a polymerase-template complex in a solutioncomprising a low concentration of nucleotides as described herein andhaving a high temperature as described herein. The solution can also besaturated with polymerase. The processivity of the polymerase-templatecomplex formed in the high-temperature solution and low nucleotidesolution is greater than a processivity resulting from a controlpolymerase-template complex solution at room temperature. For example,using the methods and compositions described herein, processivity may beincreased by about 10%, 15%, 20%, 20%, 25%, 30%, 35%, 40%, 45%, 50% ormore compared to a control that lacks low nucleotides and/or that is ranat or below room temperature.

Template Polynucleotides

The methods and compositions provided herein are applicable to variousdifferent kinds of nucleic acid templates, nascent strands, anddouble-stranded products, including single-stranded DNA; double-strandedDNA; single-stranded RNA; double-stranded RNA; DNA-RNA hybrids; nucleicacids comprising modified, missing, unnatural, synthetic, and/or rarenucleosides; and derivatives, mimetics, and/or combinations thereof.

The template nucleic acids of the invention can comprise any suitablepolynucleotide, including double-stranded DNA, single-stranded DNA,single-stranded DNA hairpins, DNA/RNA hybrids, RNAs with a recognitionsite for binding of the polymerizing agent, and RNA hairpins. Further,target polynucleotides may be a specific portion of a genome of a cell,such as an intron, regulatory region, allele, variant or mutation; thewhole genome; or any portion thereof. In other embodiments, the targetpolynucleotides may be, or be derived from mRNA, tRNA, rRNA, ribozymes,antisense RNA or RNAi.

The template nucleic acids of the invention can include unnaturalnucleic acids such as PNAs, modified oligonucleotides (e.g.,oligonucleotides comprising nucleotides that are not typical tobiological RNA or DNA, such as 240-O-methylated oligonucleotides),modified phosphate backbones and the like. A nucleic acid can be e.g.,single-stranded or double-stranded.

The nucleic acids used to produce the template nucleic acids in themethods herein (the target nucleic acids) may be essentially any type ofnucleic acid amendable to the methods presented herein. In some cases,the target nucleic acid itself comprises the fragments that can be useddirectly as the template nucleic acid. Typically, the target nucleicacid will be fragmented and further treated (e.g. ligated with adaptorsand or circularized) for use as templates. For example, a target nucleicacid may be DNA (e.g., genomic DNA, mtDNA, etc.), RNA (e.g., mRNA,siRNA, etc.), cDNA, peptide nucleic acid (PNA), amplified nucleic acid(e.g., via PCR, LCR, or whole genome amplification (WGA)), nucleic acidsubjected to fragmentation and/or ligation modifications, whole genomicDNA or RNA, or derivatives thereof (e.g., chemically modified, labeled,recoded, protein-bound or otherwise altered).

The template nucleic acid may be linear, circular (including templatesfor circular redundant sequencing (CRS)), single- or double-stranded,and/or double-stranded with single-stranded regions (e.g., stem- andloop-structures). The template nucleic acid may be purified or isolatedfrom an environmental sample (e.g., ocean water, ice core, soil sample,etc.), a cultured sample (e.g., a primary cell culture or cell line),samples infected with a pathogen (e.g., a virus or bacterium), a tissueor biopsy sample, a forensic sample, a blood sample, or another samplefrom an organism, e.g., animal, plant, bacteria, fungus, virus, etc.Such samples may contain a variety of other components, such asproteins, lipids, and non-target nucleic acids. In certain embodiments,the template nucleic acid is a complete genomic sample from an organism.In other embodiments, the template nucleic acid is total RNA extractedfrom a biological sample or a cDNA library.

In addition to increasing the processivity of the polymerase-templatecomplex at concentrations of high salt, it is contemplated that themethods and compositions provided herein can be used to offset thenegative effects on the formation of polymerase-template complexresulting from sub-optimal concentrations of cofactors, sub-optimal pHlevels and/or temperatures, or that include the presence of chemical orbiological inhibitors other than the requisite nucleotides required forpolynucleotide synthesis. For example, the polymerase-template complexcan be formed at a suboptimal pH and/or temperature in the absence ofnucleotides.

The polymerase-template complex prepared according to the methodsprovided herein can be utilized in template-dependent DNA synthesismethods including DNA amplification, and template-dependent DNAsequencing.

In some embodiments, a method is provided for performingtemplate-dependent DNA synthesis comprising (a) providing apolymerase-template complex in a solution comprising a highconcentration of salt and being essentially free of nucleotides; and (b)initiating the template-dependent DNA synthesis by adding nucleotides tothe solution. In other example embodiments, a method is provided forperforming template-dependent DNA synthesis that includes (a) providinga polymerase-template complex in a solution comprising a low nucleotideconcentration, the solution being at a high temperature, and (b)thereafter initiating the template-dependent DNA synthesis by addingnucleotides to the solution.

The polymerase of the polymerase-template complex can be a wild-type ora variant polymerase that retains polymerase activity at highconcentration of salt and/or high temperature. Examples of polymerasesthat find use in the compositions and methods described herein includethe salt-tolerant and temperature-tolerant polymerases describedelsewhere herein. In some embodiments, the polymerase of thepolymerase-template complex is a Pol6 polymerase that has an amino acidsequence that is at least 70% identical to SEQ ID NO:2. In someembodiments, the high concentration of salt is greater than 100 mM e.g.greater than 100 mM K-glu.

As described in reference to FIG. 1, processivity of the polymerase canbe increased by increasing the static processivity of the complexedpolymerase and/or increasing the replicative processivity of thecomplexed polymerase. In some embodiments, a method is provided forincreasing the static processivity of a polymerase-template complex byforming a polymerase-template complex in a solution comprising a highconcentration of salt and being essentially free of nucleotides. Theincrease in processivity of the polymerase-template complex whenprepared in the presence of a high concentration of salt in the absenceof nucleotides is greater than the processivity of thepolymerase-template complex when prepared in the same high concentrationof salt and in the presence of nucleotides. In some embodiments, theprocessivity is increased by a faster rate of association of polymerasewith template, and/or by a slower rate of dissociation of the polymerasefrom the template. The high concentration of salt can be greater than100 mM e.g. greater than 100 mM K-glu.

In some embodiments, a method is provided for increasing the staticprocessivity of a polymerase-template complex by forming apolymerase-template complex in a solution comprising a low concentrationof nucleotides and a high temperature. Additionally, the solution may besaturated with polymerase as described herein. The increase inprocessivity of the polymerase-template complex when prepared in such asolution is greater than the processivity of the polymerase-templatecomplex when prepared in a solution at room temperature and containinghigh concentrations of nucleotides.

Nanopore Sequencing Complexes—Attachment of Polymerase to Nanopore

Nanopore sequencing with the aid of a polymerase is accomplished bynanopore sequencing complexes, which are formed by linking thepolymerase-template complex to a nanopore. In some embodiments, thepolymerase-template complex is subsequently linked to a nanopore to formthe nanopore sequencing complex, which is subsequently inserted into alipid bilayer. In other embodiments, the nanopore is first inserted intoa lipid bilayer, and the polymerase-template complex is subsequentlyattached to the nanopore. Methods for assembling nanopore sequencingcomplexes are described in U.S. Provisional Application No. 62/281,719filed on Jan. 21, 2016, titled “Nanopore Sequencing Complexes,” which isincorporated herein by reference in its entirety.

Measurements of ionic current flow through a nanopore are made across ananopore that has been reconstituted into a lipid membrane. In someinstances, the nanopore is inserted in the membrane (e.g., byelectroporation, by diffusion). The nanopore can be inserted by astimulus signal such as electrical stimulus, pressure stimulus, liquidflow stimulus, gas bubble stimulus, sonication, sound, vibration, or anycombination thereof. In some cases, the membrane is formed with aid of abubble and the nanopore is inserted in the membrane with aid of anelectrical stimulus. In other embodiments, the nanopore inserts itselfinto the membrane. Methods for assembling a lipid bilayer, forming ananopore in a lipid bilayer, and sequencing nucleic acid molecules canbe found in PCT Patent Publication Nos. WO2011/097028 and WO2015/061510,which are incorporated herein by reference in their entirety.

The polymerase-template complex can be attached to the nanopore beforethe nanopore being inserted into the lipid membrane or following theinsertion of the nanopore into the lipid membrane. In certain exampleembodiments, the polymerase is attached to the nanopore, such as to oneor more of monomers of alpha-hemolysin, and the template is addedthereafter to form the polymerase-template complex.

The nanopores of the nanopore sequencing complex include withoutlimitation biological nanopores, solid state nanopores, and hybridbiological-solid state nanopores. Biological nanopores of the Pol6nanopore sequencing complexes include OmpG from E. coli, sp., Salmonellasp., Shigella sp., and Pseudomonas sp., and alpha hemolysin from S.aureus sp., MspA from M. smegmatis sp. The nanopores may be wild-typenanopores, variant nanopores, or modified variant nanopores.

Variant nanopores can be engineered to possess characteristics that arealtered relative to those of the parental enzyme. See, for example, U.S.patent application Ser. No. 14/924,861 filed Oct. 28, 2015, entitled“alpha-Hemolysin Variants with Altered Characteristics,” which isincorporated herein by reference in its entirety.

Other variant nanopores are described, for example, in U.S. ProvisionalPatent Application No. 62/357,230, filed on Jun. 30, 2016, titled “LongLifetime Alpha-Hemolysin Nanopores,” which is incorporated herein byreference in its entirety. In other example embodiments, thealpha-hemolysins of an alpha-hemolysin nanopore may be modified asdescribed in U.S. Provisional Patent Application No. 62/316,236, filedon Mar. 31, 2016, titled “Nanopore Protein Conjugates and Uses Thereof,”which is incorporated herein by reference in its entirety.

In some example embodiments, the characteristics are altered relative tothe wild-type enzyme. In some embodiments, the variant nanopore of thenanopore sequencing complex is engineered to reduce the ionic currentnoise of the parental nanopore from which it is derived. An example of avariant nanopore having an altered characteristic is the OmpG nanoporehaving one or more mutations at the constriction site (U.S. ProvisionalPatent Application No. 62/222,197, entitled “OmpG Variants”, filed onSep. 22, 2015, which is incorporated by reference herein in itsentirety), which decrease the ionic noise level relative to that of theparent OmpG. The reduced ionic current noise provides for the use ofthese OmpG nanopore variants in single molecule sensing ofpolynucleotides and proteins. In other embodiments, the variant OmpGpolypeptide can be further mutated to bind molecular adapters, whichwhile resident in the pore slow the movement of analytes, e.g.,nucleotide bases, through the pore and consequently improve the accuracyof the identification of the analyte (Astier et al., J Am Chem Soc10.1021/ja057123+, published online on Dec. 30, 2005).

Modified variant nanopores are typically multimeric nanopores whosesubunits have been engineered to affect inter-subunit interaction (U.S.Provisional Patent Application Nos. 62/232,175 and 62/244,852, entitled“Alpha-Hemolysin Variants”, filed on Sep. 24, 2015 and Oct. 22, 2015,respectively, which are incorporated by reference herein in theirentirety). Altered subunit interactions can be exploited to specify thesequence and order with which monomers oligomerize to form themultimeric nanopore in a lipid bilayer. This technique provides controlof the stoichiometry of the subunits that form the nanopore. An exampleof a multimeric nanopore whose subunits can be modified to determine thesequence of interaction of subunits during oligomerization is an aHLnanopore.

In some example embodiments, a single polymerase is attached to eachnanopore. In other embodiments, two or more polymerases are attached toa monomeric nanopore or to a subunit of an oligomeric nanopore.

Means of Attaching

The polymerase-template complex, such as the Pol6-DNA template complex,can be attached to the nanopore in any suitable way. Attachingpolymerase-polymer complexes to nanopores may be achieved using theSpyTag/SpyCatcher peptide system (Zakeri et al. PNAS 109:E690-E697[2012]) native chemical ligation (Thapa at al., Molecules 19:14461-14483[2014]), sortase system (Wu and Guo, J Carbohydr Chem 31:48-66 [2012];Heck et al., Appl Microbiol Biotechnol 97:461-475 [2013]),transglutaminase systems (Dennler et al., Bioconjug Chem 25:569-578[2014]), formylglycine linkage (Rashidian et al., Bioconjug Chem24:1277-1294 [2013]), or other chemical ligation techniques known in theart.

The polymerase-template complex can be attached to the nanopore bylinking the polymerase portion of the complex to the nanopore. In someinstances, the polymerase e.g. variant Pol6 polymerase, is linked to thenanopore using Solulink™ chemistry. Solulink™ can be a reaction betweenHyNic (6-hydrazino-nicotinic acid, an aromatic hydrazine) and 4FB(4-formylbenzoate, an aromatic aldehyde). In some instances, thepolymerase is linked to the nanopore using Click chemistry (availablefrom LifeTechnologies, for example).

In some cases, zinc finger mutations are introduced into the nanoporemolecule and then a molecule is used (e.g., a DNA intermediate molecule)to link the Pol6 polymerase to the zinc finger sites on the nanoporee.g. α-hemolysin.

Additionally, polymerase-template complex e.g. Pol6-DNA template complexcan be attached to a nanopore, e.g., aHL, OmpG, by means of a linkermolecule that is attached to a nanopore at an attachment site. In somecases, polymerase-template complex e.g. Pol6-DNA template complex, isattached to the nanopore with molecular staples. In some instances,molecular staples comprise three amino acid sequences (denoted linkersA, B and C). Linker A can extend from a nanopore monomer, Linker B canextend from the polymerase alone or from the polymerase of thepolymerase-DNA complex, and Linker C then can bind Linkers A and B(e.g., by wrapping around both Linkers A and B) and thus linking thepolymerase-template complex e.g. Pol6-DNA template complex, to thenanopore. Linker C can also be constructed to be part of Linker A orLinker B, thus reducing the number of linker molecules.

Other linkers that may find use in attaching the variant Pol6 polymeraseto a nanopore are direct genetic linkage (e.g., (GGGGS)₁₋₃ amino acidlinker (SEQ ID NO: 19)), transglutaminase mediated linking (e.g., RSKLG(SEQ ID NO: 20)), sortase mediated linking, and chemical linking throughcysteine modifications. Specific linkers contemplated as useful hereinare (GGGGS)₁₋₃ (SEQ ID NO: 19), K-tag (RSKLG (SEQ ID NO: 20)) onN-terminus, ATEV site (12-25), ATEV site+N-terminus of SpyCatcher(12-49).

An exemplary method for attaching a polymerase-template complex e.g.Pol6-DNA template complex, to a nanopore in a membrane involvesattaching a linker molecule to a nanopore or mutating a nanopore to havean attachment site and then attaching a polymerase-polynucleotidecomplex to the attachment site or attachment linker. Thepolymerase-polynucleotide complex is attached to the attachment site orattachment linker after the nanopore is inserted in the membrane. Insome cases, a polymerase-polynucleotide complex is attached to each of aplurality of nanopores that are inserted into a membrane and disposedover wells and/or electrodes of a biochip.

In some embodiments, the polymerase of the polymerase-template complexis expressed as a fusion protein that comprises a linker peptide. Thepolymerase of the polymerase-template complex can be expressed as afusion protein that comprises a SpyCatcher polypeptide, which can becovalently bound to a nanopore that comprises a SpyTag peptide (Zakeriet al. PNAS 109:E690-E697 [2012]).

A polymerase-template complex e.g. Pol6-DNA template complex, may beattached to a nanopore using methods described, for example, inPCT/US2013/068967 (published as WO2014/074727; Genia Technologies,Inc.), PCT/US2005/009702 (published as WO2006/028508; President andFellows of Harvard College), and PCT/US2011/065640 (published asWO2012/083249; Columbia University).

Biochips

Nanopores each comprising one or more polymerase-template complexprepared as described herein may be inserted in a membrane, e.g. a lipidbilayer, and disposed adjacent or in proximity to a sensing electrode ofa sensing circuit, such as an integrated circuit of a nanopore basedsensor, e.g., a biochip. The nanopore may be inserted in a membrane anddisposed of a well and/or sensing electrodes in the biochip. Multiplenanopore sensors may be provided as arrays. Biochips and methods formaking biochips are described in PCT/US20141061854 (published asWO2015/061511, Genia Technologies, Inc.), which is herein incorporatedby reference in its entirety.

The biochip can comprise nanopores each having a polymerase havingincreased processivity relative to the parental Pol6. The variant Pol6can include any of the modifications/substitutions described herein. Forexample, the variant polymerase may include a modification at one ormore amino acid residues corresponding to amino acid residues V173,N175, N176, N177, I178, V179, Y180, S211, Y212, I214, Y338, T339, G340,G341, T343, H344, A345, D417, I418, F419, K420, I421, G422, G434, A436,Y441, G559, T560, Q662, N563, E566, E565, D568, L569, I570, M571, D572,N574, G575, L576, L577, T578, F579, T580, G581, S582, V583, T584, Y596,E587, G588, E590, F591, V667, L668, G669, Q670, L685, C687, C688, G689,L690, P691, S692, A694, L708, G709, Q717, R718, V721, I734, I737, M738,F739, D693, L731, F732, T733, T287, G288, M289, R290, T291, A292, S293,S294, I295, Y342, V436, S437, G438, Q439, E440, E585, T529M, S366A,A547F, N545L, Y225L, and D657R of SEQ ID NO:2.

In some example embodiments, the modification is a substitution to aminoacid K, R, H, Y, F, W, and/or T. In some embodiments, the substitutionis a substitution to K. In some embodiments, the variant Pol6 comprisesthe substitution E585K. In other embodiments, the variant Pol6 comprisesthe substitution of two amino acids E585K+L731K. In yet otherembodiments, the variant Pol6 comprises the substitution of two aminoacids E585K+L731K. In other example embodiments, the variant Pol6 mayinclude one or more substitutions at T529M, S366A, A547F, N545L, Y225L,and/or D657R or combinations thereof. For example, the polymerasevariant may include each of the T529M+S366A+A547F+N545L+Y225L+D657Rsubstitutions. In certain example embodiments, the amino acidsubstitutions can be made in a parental Pol6 polymerase that comprises aHis6 tag (SEQ ID NO: 9) and a SpyCatcher peptide as given in thepolymerase of SEQ ID NO:4.

In certain example embodiments, the resulting variant Pol6 polymeraseshave increased processivity relative to their parental Pol6 polymerase.In some embodiments, the variant Pol6 polymerases have increasedprocessivity at a high salt concentration. In some embodiments, theincreased processivity is retained at a high salt concentration of 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800 mM or grater. In some embodiments, theincrease in processivity is displayed at a high slat concentration ofgreater than 100 mM. The increase in processivity comprises a decreasein the rate of template dissociation that is at least 2-fold less thatof the parent Pol6. Modifications of the parent Pol6 can produce variantPol6 polymerases having a rate of dissociation from the template that isat least 3-fold less that of the parent Pol6, at least 4-fold less thatof the parent Pol6, at least 5-fold less that of the parent Pol6, atleast 6-fold less that of the parent Pol6, at least 7-fold less that ofthe parent Pol6, at least 8-fold less that of the parent Pol6, at least9-fold less that of the parent Pol6, at least 10-fold less that of theparent Pol6. In some embodiments, the variant Pol6 polymerases haveincreased processivity at low nucleotide concentrations and at hightemperatures. In certain example embodiments, the polymerase hasincreased processivity at high temperatures, such as above roomtemperature as described herein.

For embodiments that include an array of nanopores in a membrane, e.g.,lipid bilayer, the density of sequencing nanopore complexes can be high.High density arrays are characterized as having a membrane surface thathas a density of Pol6 nanopore sequencing complexes greater or equal toabout to about 500 nanopore sequencing complexes per 1 mm². In someembodiments, the surface has a density of discrete nanopore sequencingcomplexes of about 100, about 200, about 300, about 400, about 500,about 600, about 700, about 800, about 900, about 1000, about 2000,about 3000, about 4000, about 5000, about 6000, about 7000, about 8000,about 9000, about 10000, about 20000, about 40000, about 60000, about80000, about 100000, or about 500000 nanopore sequencing complexes per 1mm². In some embodiments, the surface has a density of discrete nanoporesequencing complexes of at least about 200, at least about 300, at leastabout 400, at least about 500, at least about 600, at least about 700,at least about 800, at least about 900, at least about 1000, at leastabout 2000, at least about 3000, at least about 4000, at least about5000, at least about 6000, at least about 7000, at least about 8000, atleast about 9000, at least about 10000, at least about 20000, at leastabout 40000, at least about 60000, at least about 80000, at least about100000, or at least about 500000 nanopore sequencing complexes per 1mm².

The nanopore sequencing methods provided herein involve the measuring ofa current passing through the pore during interaction with thenucleotide. In some embodiments, sequencing a nucleic acid molecule canrequire applying a direct current (e.g., so that the direction at whichthe molecule moves through the nanopore is not reversed). However,operating a nanopore sensor for long periods of time using a directcurrent can change the composition of the electrode, unbalance the ionconcentrations across the nanopore and have other undesirable effects.Applying an alternating current (AC) waveform can avoid theseundesirable effects and have certain advantages as described below. Thenucleic acid sequencing methods described herein that utilized taggednucleotides are fully compatible with AC applied voltages and cantherefore be used to achieve said advantages.

Suitable conditions for measuring ionic currents through transmembraneprotein pores are known in the art and examples are provided herein inthe Experimental section. The method is carried out with a voltageapplied across the membrane and pore. The voltage used is typically from−400 mV to +400 mV. The voltage used is preferably in a range having alower limit selected from −400 mV, −300 mV, −200 mV, −150 mV, −100 mV,−50 mV, −20 mV and 0 mV and an upper limit independently selected from+10 mV, 420 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV.The voltage used is more preferably in the range 100 mV to 240 mV andmost preferably in the range of 160 mV to 240 mV. It is possible toincrease discrimination between different nucleotides by a pore of theinvention by using an increased applied potential. Sequencing nucleicacids using AC waveforms and tagged nucleotides is described in USPatent Publication US2014/0134616 entitled “Nucleic Acid SequencingUsing Tags”, filed on Nov. 6, 2013, which is herein incorporated byreference in its entirety. In addition to the tagged nucleotidesdescribed in US2014/0134616, sequencing can be performed usingnucleotide analogs that lack a sugar or acyclic moiety e.g. (S)-Glycerolnucleoside triphosphates (gNTPs) of the four common nucleobases:adenine, cytosine, guanine, and thymidine (Horhota at al. OrganicLetters, 8:5345-5347 [2006]).

Methods for Sequencing Polynucleotides

As described elsewhere herein, the molecules being characterized usingthe variant Pol6 polymerases of the Pol6 nanopore sequencing complexesdescribed herein can be of various types, including charged or polarmolecules such as charged or polar polymeric molecules. Specificexamples include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)molecules. The DNA can be a single-strand DNA (ssDNA) or a double-strandDNA (dsDNA) molecule. Ribonucleic acid can be reversed transcribed thensequenced.

In certain example embodiments, provided are methods for sequencingnucleic acids at high concentrations of salt using thepolymerase-template complexes prepared according to the methods providedherein i.e. at high concentrations of salt and in the absence ofnucleotides. The polymerase-template complexes are subsequently attachedto a nanopore to form a nanopore sequencing complex, which detectspolynucleotide sequences. In other example embodiments, provided aremethods for sequencing nucleic acids using the polymerase-templatecomplexes prepared according to the methods provided herein, such asforming the polymerase-template complexes using low nucleotideconcentrations, at high temperatures, and in the presence of excesspolymerase. The polymerase-template complexes are subsequently attachedto a nanopore to form a nanopore sequencing complex, which detectspolynucleotide sequences.

The nanopore sequencing complexes comprising polymerase-templatecomplexes prepared according to the compositions and methods providedherein, can be used for determining the sequence of nucleic acids athigh concentrations of salt using other nanopore sequencing platformsknown in the art that utilize enzymes in the sequencing ofpolynucleotides. Likewise, the nanopore sequencing complexes comprisingpolymerase-template complexes prepared according to the compositions andmethods provided, can be used for determining the sequence of nucleicacids at, for example, high temperatures using other nanopore sequencingplatforms known in the art that utilize enzymes in the sequencing ofpolynucleotides. For example, nanopore sequencing complexes comprisingthe polymerase-template complexes prepared according to the methodsdescribed herein can be used for sequencing nucleic acids according tothe helicase and exonuclease-based methods of Oxford Nanopore (Oxford,UK), Illumina (San Diego, Calif.), and the nanoporesequencing-by-expansion of Stratos Genomics (Seattle, Wash.).

In some example embodiments, sequencing of nucleic acids comprisespreparing nanopore sequencing complexes comprising polymerase-templatecomplexes prepared according to the methods described herein, anddetermining polynucleotide sequences at high concentrations of saltusing tagged nucleotides as is described in PCT/US2013/068967 (entitled“Nucleic Acid Sequencing Using Tags” filed on Nov. 7, 2013, which isherein incorporated by reference in its entirety). For example, ananopore sequencing complex that is situated in a membrane (e.g., alipid bilayer) adjacent to or in sensing proximity to one or moresensing electrodes, can detect the incorporation of a tagged nucleotideby a polymerase at a high concentration of salt as the nucleotide baseis incorporated into a strand that is complementary to that of thepolynucleotide associated with the polymerase, and the tag of thenucleotide is detected by the nanopore. The polymerase-template complexcan be associated with the nanopore as provided herein.

Tags of the tagged nucleotides can include chemical groups or moleculesthat are capable of being detected by a nanopore. Examples of tags usedto provide tagged nucleotides are described at least at paragraphs[0414] to [0452] of PCT/US2013/068967. Nucleotides may be incorporatedfrom a mixture of different nucleotides, e.g., a mixture of tagged dNTPswhere N is adenosine (A), cytidine (C), thymidine (T), guanosine (G) oruracil (U). Alternatively, nucleotides can be incorporated fromalternating solutions of individual tagged dNTPs, i.e., tagged dATPfollowed by tagged dCTP, followed by tagged dGTP, etc. Determination ofa polynucleotide sequence can occur as the nanopore detects the tags asthey flow through or are adjacent to the nanopore as the tags reside inthe nanopore and/or as the tags are presented to the nanopore. The tagof each tagged nucleotide can be coupled to the nucleotide base at anyposition including, but not limited to a phosphate (e.g., gammaphosphate), sugar or nitrogenous base moiety of the nucleotide. In somecases, tags are detected while tags are associated with a polymeraseduring the incorporation of nucleotide tags. The tag may continue to bedetected until the tag translocates through the nanopore afternucleotide incorporation and subsequent cleavage and/or release of thetag. In some cases, nucleotide incorporation events release tags fromthe tagged nucleotides, and the tags pass through a nanopore and aredetected. The tag can be released by the polymerase, or cleaved/releasedin any suitable manner including without limitation cleavage by anenzyme located near the polymerase. In this way, the incorporated basemay be identified (i.e., A, C, G, T or U) because a unique tag isreleased from each type of nucleotide (i.e., adenine, cytosine, guanine,thymine or uracil). In some situations, nucleotide incorporation eventsdo not release tags. In such a case, a tag coupled to an incorporatednucleotide is detected with the aid of a nanopore. In some examples, thetag can move through or in proximity to the nanopore and be detectedwith the aid of the nanopore.

Thus, in one aspect, a method is provided for sequencing apolynucleotide from a sample, e.g. a biological sample, with the aid ofa nanopore sequencing complex at a high concentration of salt. Thesample polynucleotide is combined with the polymerase in a solutioncomprising a high concentration of salt and being essentially free ofnucleotides to provide the polymerase-template complex portion of thenanopore sequencing complex. In one embodiment, the samplepolynucleotide is a sample ssDNA strand, which is combined with a DNApolymerase to provide a polymerase-DNA complex e.g. a Pol6-DNA complex.

In some embodiments, nanopore sequencing of a polynucleotide sample isperformed by providing a polymerase-template complex e.g. Pol6-templateor variant Pol6-template complex in a solution comprising a highconcentration of salt e.g. greater than 100 mM, and being essentiallyfree of nucleotides; attaching the polymerase-template complex to ananopore to form a nanopore-sequencing complex; and providingnucleotides to initiate template-dependent strand synthesis. Thenanopore portion of the sequencing complex is positioned in the membraneadjacent to or in proximity of a sensing electrode, as describedelsewhere herein. The resulting nanopore sequencing complex is capableof determining the sequence of nucleotide bases of the sample DNA at ahigh concentration of salt as described elsewhere herein. In otherembodiments, the nanopore sequencing complex determines the sequence ofdouble stranded DNA. In other embodiments, the nanopore sequencingcomplex determines the sequence of single stranded DNA. In yet otherembodiments, nanopore sequencing complex determines the sequence of RNAby sequencing the reverse transcribed product.

In some embodiments, a method is provided for nanopore sequencing at ahigh salt concentration. The method comprises (a) providing apolymerase-template complex in a solution comprising a highconcentration of salt e.g. at least 100 mM, and being free ofnucleotides; (b) combining the polymerase-template complex with ananopore to form a nanopore-sequencing complex; (c) providing taggednucleotides to the nanopore sequencing complex to initiatetemplate-dependent nanopore sequencing in a high salt concentration ofat least 100 mM salt; and (d) detecting with the aid of the nanopore, atag associated with each of the tagged nucleotides during incorporationof each of the nucleotides to determine that sequence of the template.The polymerase of the polymerase-template complex can be a wild-type ora variant polymerase that retains polymerase activity at highconcentration of salt. Examples of polymerases that find use in thecompositions and methods described herein include the salt-tolerantpolymerases described elsewhere herein. In some embodiments, thepolymerase of the polymerase-template complex is a Pol6 polymerase thathas an amino acid sequence that is at least 70% identical to SEQ IDNO:2.

In some embodiments, a method for nanopore sequencing a nucleic acidsample is provided. The method comprises using nanopore sequencingcomplexes comprising the variant Pol6 polymerases provided herein. Inone embodiment, the method comprises providing tagged nucleotides to aPol6 nanopore sequencing complex, and under high salt conditions,carrying out a polymerization reaction to incorporate the nucleotides ina template-dependent manner, and detecting the tag of each of theincorporated nucleotides to determine the sequence of the template DNA.

In one embodiment, tagged nucleotides are provided to a Pol6 nanoporesequencing complex comprising a variant Pol6 polymerase provided herein,and under conditions of high salt, carrying out a polymerizationreaction with the aid of the variant Pol6 enzyme of said nanoporesequencing complex, to incorporate tagged nucleotides into a growingstrand complementary to a single stranded nucleic acid molecule from thenucleic acid sample; and detecting, with the aid of nanopore, a tagassociated with said individual tagged nucleotide during incorporationof the individual tagged nucleotide, wherein the tag is detected withthe aid of said nanopore while the nucleotide is associated with thevariant Pol6 polymerase.

In one aspect, a method is provided for sequencing a polynucleotide froma sample, e.g. a biological sample, with the aid of a nanoporesequencing complex at a high temperature and at a low concentration ofnucleotides. For example, the sample polynucleotide is combined with thepolymerase in a solution having a high temperature and having a lowconcentration of nucleotides. In one embodiment, the samplepolynucleotide is a sample ssDNA strand, which is combined with a DNApolymerase to provide a polymerase-DNA complex e.g. a Pol6-DNA complex.The temperature may be above room temperature, such as at about 40° C.,as described herein. The nucleotide concentration, for example, may beabout 1.2 μM, as described herein. Further, the solution may include ahigh concentration of the polymerase, such as being saturated with thepolymerase. The polymerase can be a variant polymerase as describedherein.

In certain example aspects, a method is provided for nanopore-basedsequencing of a polynucleotide template. The method includes forming apolymerase-template complex, as described herein, in a solutionincluding a low concentration of nucleotides, the solution having a hightemperature, such as above room temperature. For example, thetemperature may be about 40° C., as described herein. The methodincludes combining the formed polymerase-template complex with ananopore to form a nanopore-sequencing complex. Tagged nucleotides canthen be provided to the nanopore sequencing complex to initiatetemplate-dependent nanopore sequencing of the template at the hightemperature. With the aid of the nanopore, a tag associated with each ofthe tagged nucleotides during incorporation of each of the taggednucleotides while each of the tagged nucleotides is associated with thepolymerase is detected, thereby determining the sequence of thepolynucleotide template. In certain examples, forming thepolymerase-template complex includes saturating the solution with thepolymerase of the polymerase-template complex. The nucleotideconcentration can be 0.8 μM to 2.2 μM, such as about 1.2 μM. Thetemperature, for example, can be about 35° C. to 45° C., such as about40° C.

Other embodiments of the sequencing method that comprise the use oftagged nucleotides with the present nanopore sequencing complexes forsequencing polynucleotides are provided in WO2014/074727, which isincorporated herein by reference in its entirety.

Sequencing nucleic acids using AC waveforms and tagged nucleotides isdescribed in US Patent Publication US2014/0134616 entitled “Nucleic AcidSequencing Using Tags”, filed on Nov. 6, 2013, which is hereinincorporated by reference in its entirety. In addition to the taggednucleotides described in US2014/0134616, sequencing can be performedusing nucleotide analogs that lack a sugar or acyclic moiety, e.g.,(S)-Glycerol nucleoside triphosphates (gNTPs) of the five commonnucleobases: adenine, cytosine, guanine, uracil, and thymidine (Horhotaet al. Organic Letters, 8:5345-5347 [2006]).

Reagents, Storage Solutions, and Kits

Sequencing reagents for DNA sequencing or amplification e.g. nanoporesequencing are also provided, the reagent(s) comprising apolymerase-template complex in a solution comprising a highconcentration of salt and being essentially free of nucleotides. Incertain example embodiments, the reagent(s) include a polymerase andtemplate in a solution with low levels of nucleotides, where thesolution can be warmed to a high temperature as described herein toinitiate and/or enhance formation of the polymerase-template complex. Insuch embodiments, the solution can be saturated with polymerase. In someembodiments, the polymerase of the polymerase-template complex comprisesa polymerase that is a wild-type or a variant polymerase that retainspolymerase activity at high concentration of salt e.g. Pol6 of any oneof SEQ ID NOs: 1, 2, 4, 6, 7, 8 and 14. Examples of polymerases thatfind use in the compositions and methods described herein include thesalt-tolerant and/or high-temperature tolerant polymerases describedelsewhere herein. In some embodiments, the polymerase of thepolymerase-template complex is a Pol6 polymerase that has an amino acidsequence that is at least 70% identical to SEQ ID NO:2.

In some embodiments, the polymerase of the polymerase-template complexis a Pol6 polymerase that has an amino acid sequence having at least 70%identity to full-length parent polypeptide of SEQ ID NO:2 and comprisesone or more amino acid substitutions of amino acid residuescorresponding to amino acids V173, N175, N176, N177, I178, V179, Y180,S211, Y212, I214, Y338, T339, G340, G341, T343, H344, A345, D417, I418,F419, K420, I421, G422, G434, A436, Y441, G559, T560, Q662, N563, E565,E566, D568, L569, I570, M571, D572, N574, G575, L576, L577, T578, F579,T580, G581, S582, V583, T584, Y596, E587, G588, E590, F591, V667, L668,G669, Q670, L685, C687, C688, G689, L690, P691, S692, A694, L708, G709,Q717, R718, V721, I734, I737, M738, F739, D693, L731, F732, T733, T287,G288, M289, R290, T291, A292, S293, S294, I295, Y342, V436, S437, G438,Q439, E440, and E585, T529M, S366A, A547F, N545L, Y225L, and D657R ofSEQ ID NO:2. In some embodiments, the amino acids substitution(s) is toK, R, Y, F, W, and/or T. In some embodiments, the sequencing reagentcomprises the variant Pol6 polymerase of SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, and/or SEQ ID NO: 14. In some embodiments, the sequencingreagent comprises a polynucleotide encoding any one of the variant salttolerant or heat-tolerant Pol6 polymerases provided herein.

In another example embodiment, a storage solution is provided. Thestorage solution comprises a polymerase-template complex in a solutioncomprising a high concentration of salt. In some embodiments, the highconcentration of salt is greater than 100 mM salt e.g. greater than 100mM K-glu. In another example embodiment, the storage solution includes apolymerase and template in a solution with low levels of nucleotides,where the solution can be warmed to a high temperature as describedherein to initiate and/or enhance formation of the polymerase-templatecomplex. The storage solution, for example, can be saturated withpolymerase.

In another aspect, a kit including a sequencing reagent for DNAsequencing is provided. In some embodiments, the kit comprises apolymerase-template complex in a solution comprising a highconcentration of salt and being free of nucleotides. In someembodiments, the kit further comprises a buffer and/or nucleotides. Icertain example embodiments, the kit includes a polymerase and templatein a solution with low levels of nucleotides, where the solution can bewarmed to a high temperature as described herein to initiate and/orenhance formation of the polymerase-template complex. In suchembodiments, the solution can be saturated with polymerase. The solutionof the kit may also include a buffer. The polymerase of the kits can,for example, be a wild type polymerase or a variant polymerase, such anyof the variant polymerases described herein.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); kg (kilograms); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); h (hours); min (minutes); sec (seconds); msec(milliseconds).

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and scope of the appended claims. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

Example 1: Directed Mutagenesis Pol6 Mutants

DNA of SEQ ID NO: 3 encoding the WT-Pol6 (SEQ ID NO: 2) was purchasedfrom a commercial source (DNA 2.0, Menlo Park, Calif.). The sequence wasverified by sequencing.

Site directed mutagenesis was performed to mutate one or more aminoacids of the putative nucleotide/DNA binding site of parental variantPol6-44-X1 (SEQ ID NO:4). Pol6-44-X1 was derived from wild-type Pol6 tocomprise the following substitutions: S366A T529M A547F D44A (SEQ IDNO:4). Pol6-67-X2 was derived from wild-type Pol6 to comprise thefollowing mutations: S366A T529M A547F N545L Y225L D657R Y242A (see SEQID NO: 14).

The Pol6 variants like 44-X1 were expressed as a fusion protein havingan N-terminal His-tag (see underlined sequence in SEQ ID NO:4) andSpyCatcher domain (bolded italic sequence in SEQ ID NO:4).

Mutagenesis Protocol

The primers for each mutagenesis reaction were designed using the NEBbase changer protocol and ordered in 96-well plate format from IDT.

The forward and reverse primes were 5′ phosphorylated in high throughput(HTP) format using the T4 polynucleotide kinase (PNK) purchased fromNEB. A typical 25-μl reaction contained 15 μl of primer at 10 μM, 5 μlof 5× reaction buffer (from NEB), 1.25 μl PNK enzyme, 3.75 μl water. Thereaction was performed at 37° C. for 30 min and the enzyme heatinactivated at 65° C. for 20 min.

PCR mutagenesis was performed using Q5 DNA polymerase from NEB. Atypical 25 μl reaction contained 5 μl of Q5 buffer, 5 μl of GC enhancer,0.5 ul of 10 mM dNTPs, 1.25 μl of 10 μM phosphorylated mutagenesisprimers forward and reverse, 0.25 μl Q5 polymerase and 1 μl of 5 ng/mlwild type Pol6 template, i.e., His-Pol6, and 10.75 μl H₂0.

Once PCR was completed, 0.5 μl of Dpn1 was added to 25 μl PCR mix andincubated at 37° C. for 1 hr. Then, 2.5 μl of Blunt/TA ligase master mixwere added to 2.5 μl of Dpn1 treated PCR product, and the reactionmixture was incubated at room temperature for 1 hr. Thereafter, 1 μl ofligation mix was added to 20 ul of 96-well BL21DE3 cells (EMD Millipore)and incubated on ice for 5 min.

The cells were heat shocked at 42° C. for exactly 30 sec using the PCRthermocycler and placed on ice for 2 min. Thereafter, 80 μl of SOC wereadded to the cells, which were then incubated at 37° C. for 1 hr withoutshaking. A 100 μl aliquot of SOC or ultra-pure water were added to thecells, which were then plated on 48-well LB-agar plates comprising50-100 μg/ml kanamycin. Cells were grown overnight at 37 C.

Example 2: Expression and Purification

Variants of the parental polymerase Pol6-44-X1 (SEQ ID NO:4), and thePol6-67-X2 (SEQ ID NO: 14) were expressed and purified using a highthroughput method as follows.

DNA encoding variants in expression plasmid pD441 vector weretransformed into competent E. coli, and glycerol stocks of thetransformed cells were made. Starting from a tiny pick of the glycerolstock, grow 1 ml starter culture in LB with 0.2% Glucose and 100 μg/mlKanamycin for approximately 8 hrs. Transfer 25 μl of log phase starterculture into 1 ml of expression media (Terrific Broth (TB) autoinductionmedia supplemented with 0.2% glucose, 50 mM Potassium Phosphate, 5 mMMgCl2 and 100 μg/ml Kanamycin) in 96-deep well plates. The plates wereincubated with shaking at 250-300 rpm for 36-40 hrs at 28° C.

Cells were then harvested via centrifugation at 3200×g for 30 minutes at4° C. The media was decanted off and the cell pellet resuspended in 200μl pre-chilled lysis buffer (20 mM Potassium Phosphate pH 7.5, 100 mMNaCl, 0.5% Tween20, 5 mM TCEP, 10 mM Imidazole, 1 mM PMSF, 1× BugBuster, 100 μg/ml Lysozyme and protease inhibitors) and incubated atroom temperature for 20 min with mild agitation. Then, 20 μl was addedfrom a 10× stock to a final concentration of 100 μg/ml DNase, 5 mMMgCl2, 100 μg/ml RNase I and incubated in on ice for 5-10 min to producea lysate. The lysate was supplemented with 200 μl of 1M PotassiumPhosphate, pH 7.5 (Final concentration will be about 0.5M Potassiumphosphate in 400 μl lysate) and filtered through Pall filter plates(Part#5053, 3 micron filters) via centrifugation at approximately 1500rpm at 4 C for 10 minutes. The clarified lysates were then applied toequilibrated 96-well His-Pur Cobalt plates (Pierce Part#90095) and bindfor 15-30 min.

The flow through (FT) was collected by centrifugation at 500×G for 3min. The FT was then washed 3 times with 400 ul of wash buffer 1 (0.5MPotassium Phosphate pH 7.5, M NaCl 5 mM TCEP, 20 mM Imidazole+0.5%Tween20). The FT was then washed twice in 400 ul wash buffer 2 (50 mMTris pH 7.4, 200 mM KCl, 5 mM TCEP, 0.5% Tween20, 20 mM Imidazole).

The Pol6 was eluted using 200 μl elution buffer (50 mM Tris Ph7.4, 200mM KCl, 5 mM TCEP, 0.5% Tween20, 300 mM Imidazole, 25% Glycerol) andcollected after 1-2 min incubation. Reapply eluate to the same His-Purplate2-3 times to get concentrated Pol6 in elute. The purifiedpolymerase is >95% pure as evaluated by SDS-PAGE. The proteinconcentration is ˜3 uM (0.35 mg/ml) with a 260/280 ratio of 0.6 asevaluated by Nanodrop.

Example 3: Template Association Experiments

The association of polymerase-template complex was assayed using theShortCy5Template (/5Cy5/AGA GTG ATA GTA TGA TTA TGT AGA TGT AGG ATT TGATAT GTG AGT AGC CGA ATG AAA CCT T/iSpC3/TT GGT TTC ATT CGG) (SEQ ID NOS12 and 21) and the ShortBHQ2Primer (TTT TCA TAA TCA TAC TAT CACTCT/BHQ2/-3) (SEQ ID NO: 13).

The association of polymerase-template complex was assayed under thefollowing conditions: (A) 2× Pol6-44X1 polymerase (SEQ ID NO:4) waspre-incubated with 50 nM ShortCy5Template (SEQ ID NOS 12 and 21) in thepresence of Mg²⁺ alone for 32, 55, and 85 minutes at which timespolynucleotide synthesis was initiated by adding polyphosphatenucleotides; (B) 2× Pol6-44X1 polymerase (SEQ ID NO:4) was pre-incubatedwith 50 nM ShortCy5Template (SEQ ID NOS 12 and 21) in the presence ofpolyphosphate nucleotides alone for 32, 55, and 85 minutes at whichtimes polynucleotide synthesis was initiated by adding MgCl2; or (C) 2×Pol6-44X1 polymerase (SEQ ID NO:4) was pre-incubated with 50 nMShortCy5Template (SEQ ID NOS 12 and 21) in the absence of MgCl2 andpolyphosphate nucleotides alone for 32, 55, and 85 minutes at whichtimes polynucleotide synthesis was initiated by adding MgCl2 andpolyphosphate nucleotides.

The level of polymerase-template complex formation was measured for eachof the three assay conditions at increasing concentration of K-glu: 75mMK-glu, 150 mM K-glu, and 300 mM K-glu.

The fluorescence in each case was measured after the reactions wereinitiated using excitation at 648 nm (590-50) nm and emission at 668 nm(675-50) and were measured every 0.1 s for 1 min.

The results are shown in FIGS. 3 (A-C) and corresponding FIGS. 4 A-C.More particularly, FIG. 3 shows the fluorescence signal obtained at 32minutes, 55 minutes and 85 minutes for each of the assay conditionsdescribed above. The amplitude of the signal (in RFU) represents thelevel of DNA-Pol6 complex. Numerical values for the signals' amplitudewere calculated and represented in corresponding FIG. 4 (A-C), whereFIGS. 4A, 4B, and 4C show the amplitude of fluorescence signal obtainedunder assay condition A, B, and C, respectively. Diamonds (□) representsignal amplitude measured at 75 mM K-glu, (□) represent signal amplitudemeasured at 150 mM K-glu, and triangles (Δ) represent signal amplitudemeasured at 300 mM K-glu.

The data shown in FIGS. 3 and 4 demonstrate that the level ofpolymerase-template complex formed at 75 mM and 150 mM K-glu isindependent of the incubation conditions i.e. pre-incubation of DNA withPol6 in the presence of Mg₂ ⁺, alone or when in combination withnucleotides did not affect template binding to Pol6. However, at a highsalt concentration of 300 mM K-glu, the binding of DNA to Pol6 wasdiminished when the complex was allowed to form in the presence ofnucleotides alone. The same effect was also seen at a salt concentrationof 500 mM K-glu (data not shown).

These data indicate that at high salt concentrations, nucleotidesinterfere with the binding of DNA template to polymerase, and therebydecrease the level of polymerase-template complex.

Example 4: Template Dissociation Experiments

This example (4.1-4.5) demonstrates the effect of nucleotides on thedissociation of template from the template-polymerase complex.

The effect of divalent metal ions i.e. Mg₂ ⁺, and/or nucleotides wasdetermined on the rate of dissociation of template from apolymerase-template complex (koff) at high salt concentration e.g. 500mM K-glu as follows. Polymerase-template complex was allowed to form inthe presence of 75 mM K-glu. At time=0, the concentration of salt wasraised to 500 mM, and the subsequent dissociation of the complex wasdetermined at 15, 30, 45, 60, 75, 90 120, 150, 180, 210, and 240 minutesby initializing polynucleotide synthesis under the following five assayconditions according to the FRET assay described in Example 3.

4.1. Blocked Nucleotides Inhibit Formation of Polymerase-TemplateComplex

2× concentration of Pol6-44X1 (SEQ ID NO:4) was pre-incubated withShortCy5Template (SEQ ID NOS 12 and 21) in the presence of 5 mM MgCl2(A(i)) or in the presence of 5 mM MgCl2+0.1 μM dnpCpp (blockednucleotide) (A(ii)) at a salt concentration of 75 mM K-glu to allow forthe formation of template-DNA complex. At time=0 minutes, salt was addedto a final concentration of 500 mM KGlu. Dissociation of template fromthe template-DNA complex was determined following addition ofpolyphosphates at various time intervals.

FIG. 5A shows the fluorescence signal corresponding to the level ofpolymerase-template complex detected under conditions given in A(i) andA(ii).

FIG. 5B shows a plot of the dissociation of polymerase from thepolymerase-template complex when the complex was allowed to form in thepresence of Mg²⁺ (♦), or in the presence of 5 mM Mg²⁺+0.1 μM dnpCpp (▪).The calculated amplitude of the fluorescence signal shown in 5A (i) and(ii) is plotted in RFU as a function of time.

The data show that blocked nucleotides inhibit binding of template topolymerase.

4.2. Formation of Polymerase-Template Complex in the Presence ofNucleotides Increases the Rate of Template Dissociation from PolymeraseOver Time.

2× concentration of Pol6-44X1 (SEQ ID NO:4) was pre-incubated withShortCy5Template (SEQ ID NOS 12 and 21). Binding was allowed to proceedin the presence of 5 mM MgCl₂, followed by addition of 20 μMpolyphosphate nucleotides nucleotides (FIG. 6A(i)) to initiate thereaction; or binding occurred in the presence of 50 uM polyphosphatenucleotides polyphosphates, followed by addition of Mg² (FIG. 6A(ii)) toinitiate the reaction (note the final concentration of polyphosphates is20 uM in both cases). At time=0 minutes, salt was added to a finalconcentration of 500 mM KGlu. Dissociation of template from thetemplate-DNA complex was determined following addition of polyphosphates(6A(i)) or MgCl2 (6A(ii)) at various time intervals.

The data are shown in FIG. 6A (i) and (ii), and FIG. 6B. Moreparticularly, FIG. 6A shows the fluorescence signal corresponding to thelevel of polymerase-template complex detected under conditions given in6A(i) and 6A(ii). FIG. 6B shows a plot of the dissociation of polymerasefrom the polymerase-template complex when the complex was allowed toform in the presence of Mg²⁺ alone (♦), or in the presence ofpolyphosphate nucleotides (▪). The calculated amplitude of thefluorescence signal shown in 6A (i) and (ii) is plotted as a function oftime.

These data show that forming the polymerase-template complex in thepresence of 50 uM polyphosphates results in a greater rate of polymerasedissociation from template than when polymerase-template complex isformed in the presence of Mg2+ and the absence of polyphosphates.

4.3. Ca2+ does not Improve the Nucleotide-Dependent Destabilization i.e.Dissociation, of Polymerase-Template Complex.

2× concentration of Pol6-44X1 (SEQ ID NO:4) was pre-incubated withShortCy5Template (SEQ ID NOS 12 and 21). Binding was allowed to proceedin the presence of 50 μM polyphosphates, followed by addition of 5 mMMgCl2 (FIG. 7A(i)) to initiate the reaction; or binding occurred in thepresence of 50 μM polyphosphates+0.5 mM Ca2+, followed by addition of 5mM Mg2+(FIG. 7A(ii)) to initiate the reaction. At time=0 minutes, saltwas added to a final concentration of 500 mM KGlu. Dissociation oftemplate from the template-DNA complex was determined following additionof MgCl2 (7A) at various time intervals.

The data are shown in FIG. 7A (i) and (ii), and FIG. 7B. Moreparticularly, FIG. 7A shows the fluorescence signal corresponding to thelevel of polymerase-template complex detected under conditions given in7A(i) and 7A(ii). FIG. 7B shows a plot of the dissociation of polymerasefrom the polymerase-template complex when the complex was allowed toform in the presence of polyphosphates (♦) or in the presence ofpolyphosphates+Ca²⁺ (▪). The calculated amplitude of the fluorescencesignal shown in 7A (i) and (ii) is plotted as a function of time.

As shown in FIG. 7B, the effect of Ca²⁺ does not affect complexdissociation. FIG. 7B also shows that the rapid rate of complexdissociation following template binding in the presence of nucleotidesis similar whether occurring in the absence or presence of Ca²⁺.

4.4. Mg2+ does not Improve the Nucleotide-Dependent Destabilization i.e.Dissociation, of Polymerase-Template Complex During PolynucleotideSynthesis.

2× Pol6-44X1 polymerase (SEQ ID NO:4) was pre-incubated withShortCy5Template (SEQ ID NOS 12 and 21). Binding was allowed to proceedin the absence of Mg2+ and polyphosphates, followed by addition of Mg2+and polyphosphates to initiate the reaction (FIG. 8A(i)); in thepresence of Mg2+ followed by addition of polyphosphates to initiate thereaction (FIG. 8A(ii)); or in the presence of polyphosphates, followedby addition of Mg2+(FIG. 8A(iii)). At time=0 minutes, salt was added toa final concentration of 500 mM KGlu. Dissociation of template from thetemplate-polymerase complex was determined following the addition ofMg2+ and polyphosphates, only polyphosphates, or only Mg2+ at differenttime intervals.

The data are shown in FIG. 8A(i), 8A(ii), 8A(iii), and FIG. 8B. Moreparticularly, FIG. 8A (i)-(iii) shows the fluorescence signalcorresponding to the level of polymerase-template complex detected underconditions given in 8.4 A(i), 8.4 A(ii) and 8.4 A(iii), respectively.FIG. 8B shows a plot of the dissociation of polymerase from thepolymerase-template complex. The calculated amplitude of thefluorescence signal shown in 8A (i), (ii), and (iii) is plotted as afunction of time. The data in FIG. 8A(i) and (ii) show thatpolymerase-template complex formation is similar whether it occurred inthe presence of Mg2+(ii), or in the absence of both Mg2+ andnucleotides. The data shown in FIG. 8A(iii) show that nucleotidesinhibit template binding. FIG. 88B shows that the rate of dissociationof complex when formed in the presence of Mg2+ (▪), or in the absence ofMg2+ and nucleotides (♦) is similar. FIG. 8B (Δ) also shows thatformation of complex in the presence of polyphosphates increases therate of complex dissociation, i.e. nucleotides destabilizepolymerase-template complexes over time.

4.5. Nucleotide Triphosphates Increase the Rate of Template-PolymeraseDissociation when Compared to Polyphosphates.

2× concentration Pol6-44X1 polymerase (SEQ ID NO:4) was pre-incubatedwith DNA template, i.e., ShortCy5Template (SEQ ID NOS 12 and 21).Binding was allowed to proceed in the presence of polyphosphates,followed by addition of Mg²⁺ to initiate the reaction (FIG. 9A(i)); orin the presence of triphosphate nucleotides followed by addition of Mg²⁺to initiate polynucleotide synthesis (condition 9A(ii)). At time=0minutes, salt was added to a final concentration of 500 mM KGlu.Dissociation of template from the template-DNA complex was determinedfollowing addition of Mg2+(9A) at various time intervals.

The data are shown in FIGS. 9A(i) and 9A(ii), and FIG. 9B. Moreparticularly, FIG. 9A (i)-(ii) shows the fluorescence signalcorresponding to the level of polymerase-template complex detected underconditions given in 5.5 A(i) and 5.5 A(ii), respectively. FIG. 9B showsa plot of the dissociation of polymerase from the polymerase-templatecomplex. The calculated amplitude of the fluorescence signal shown in 9A(i) and (ii) is plotted as a function of time.

The data in FIG. 9A(i) and (ii) show that polymerase-template complexformation is similar whether it occurred in the presence of dNTP orpolyphosphate nucleotides. FIG. 9B shows that the rate of dissociationof complex when formed in the presence of dNTP nucleotides (U) isgreater than when in the presence of polyphosphate nucleotidesnucleotides (o).

In sum, the data show that formation of template-polymerase complex inthe presence of triphosphate nucleotides results in a higher rate oftemplate dissociation when compared to polyphosphate. This effect isexpected to result in lower processivity and diminished sequencinglongevity during template-dependent DNA polymerization.

Example 5: Effect of Temperature and Nucleotides on Binding ofPolymerase to Template

This example demonstrates the effect of temperature on the associationof template from the template-polymerase complex, with and without lowconcentration of nucleotides.

Varying dilutions (0×, 1×, 4×, 8×) of Pol6-67 X2 (SEQ ID NO: 14) werepre-incubated with 100 nM Fluorescent Hairpin DNA template (SEQ ID NOS15 and 22) either in the presence of 1.2 uM polyphosphate nucleotidesnucleotides at 40° C. or in the absence of 1.2 uM polyphosphatenucleotides at room temperature for 30 minutes. 12 uL of pre-boundtemplate-Pol complex was loaded onto 5% Native-TBE gel and run at 100Vfor 60 minutes at 4° C. Imaging was performed using Biorad's ChemiDocXRS+ imaging system using SYBR-Green filter.

As shown in FIG. 10A, at 20° C., the 4× and 8× polymerase concentrationsresult in band shifts, thus indicating non-specific binding of multiplepolymerases to multiple locations on the template. In contrast,increasing the temperature to 40° C. and adding 1.2 μM polyphosphatenucleotides did not result in the band shift (see FIG. 10B), thusindicating specific binding of the polymerase to the 3′ end of thetemplate. Hence, the addition of 1.2 μM polyphosphate and elevatedtemperate have a positive effect on polymerase-template binding.

Example 6: Effect of Temperature and Nucleotides on Template Extension(Extension Gel Assay)

This example demonstrates the correlation between the percent ofpolymerase bound to the template (at high temperature and low nucleotidelevels) and extension of the template (at high temperature and highconcentration of nucleotides).

Varying dilutions of Pol16-67 X2 (0×, 1×, 2×, 4×, 8×) were pre-incubatedwith 300 mM Fluorescent Hairpin DNA template (SEQ ID NOS 15 and 22) inthe presence of 1.2 uM polyphosphate nucleotides nucleotides at 40° C.for 30 minutes. The binding buffer was a Hepes buffer having 75 mMK-Glu, 20 mM Hepes (pH 7.5), 5 mM TCEP, and 8% Trehalose.

For the binding gel, 12 uL of pre-bound template-Pol complex was loadedonto 5% Native-TBE gel and run at 100V for 60 minutes at 4° C. Imagingwas performed using Biorad's ChemiDoc XRS+ imaging system usingSYBR-Green filter.

For the extension reaction, 10 uM of polyphosphate nucleotides, 5 mMMgCl2 (Final each) and 20× Chase template (SEQ ID NO: 16) was added tothe pre-bound Polymerase-template complex to initiate the reaction. Thereactions were ran for 5 mins at 30° C. After 5 minutes, the reactionswere quenched using Formamide+50 mM EDTA, and heated at 95° C. for 5minutes. 12 uL of the samples were then loaded on to 15% TB-Urea Gel at180V for 180 minutes and imaged using Biorad's ChemiDoc XRS+ imagingsystem using SYBR-Green filter.

As shown in FIG. 11A, increasing polymerase concentration results in anincrease in template binding at 40° C. in the presence of low (1.2 μM)polyphosphate nucleotides. As evidenced by the shifts in band intensityfrom the lower band to the upper band with increased concentration ofpolymerase in FIG. 11B, increasing the concentration of polymeraseresults in increased template extension, with the concentration ofnucleotides adjusted to 10 μM during the extension reaction. At 1×polymerase, the active fraction shows 26% extension, whereas at 8×polymerase the active fraction shows 66% extension at 40° C. (FIG. 11B).When percent binding is compared with percent extension, a directcorrelation exists (see FIG. 11C; slope=1). Hence, the active fractionis largely dependent on polymerase binding to the template beforeextension.

Example 7: Effect of Temperature and Nucleotides on Template Extension(Fret Assay)

This example demonstrates formation and extension of thepolymerase-template complex and template extension at 40° C. using aFRET assay (as described in Example 3).

Equi-molar quantities of LongHP-Cy5-ExoR template (SEQ ID NOS 17 and 22)were annealed with Quencher Primer (SEQ ID NO: 18) using the cool-downannealing protocol. A control was made in which only LongHP-Cy5-ExoRtemplate (SEQ ID NOS 17 and 22) (at the same final concentration) wasdiluted in 1×TE and was also passed through the cool-down annealingprotocol.

Varying dilutions of Pol6-67 X2 (0×, 1×, 2×, 4×, 6×, 8×) werepre-incubated with either 50 mM annealed Template-Primer pair or withjust the Template control in the presence of 1.2 uM polyphosphatenucleotides nucleotides at 40° C. for 30 minutes. The binding buffer wasa Hepes buffer having 75 mM K-Glu, 20 mM Hepes (pH 7.5), 5 mM TCEP, and8% Trehalose.

The above reactions were carried out in a 96 well half area blackplates. The plate reader (BMG FLUOstar Omega) injected Reagent B, thatcontained 75 mM K-Glu, 20 mM Hepes, 5 mM TCEP, 5 mM MgCl2, 10 uM Nucs,20× Chase (final concentrations), which initiated the reaction and thefluorescence was measured every 1 s for 10 minutes. The excitationfilter used is 590-50 nm and the emission filter used is 675-50.

FIGS. 12A-12C show template extension following polymerase-templateformation at 40° C. and in the presence of low levels of nucleotides(1.2 μM). As shown in FIG. 12A and FIG. 12B, followingpolymerase-template formation, increasing polymerase concentrationresults in increased extension, as evidenced the by increased signalamplitude of the fluorophore quencher at increased polymeraseconcentrations. At 0× polymerase, for example, no binding of thetemplate to the polymerase can occur and the fluorescent signal is thuscompletely quenched. Increasing the polymerase concentration duringpolymerase-template formation, however, results in less of the signalbeing quenched (which corresponds to an increase in fluoresce amplitude)(FIGS. 12A and 12B). The control fluorophore alone remains maximallyfluorescent (i.e., 100% saturation) across the various polymeraseconcentrations (FIGS. 12A and 12B). As shown in FIG. 12C, the percentextension—as determined as a percentage of the 100% saturation of thefluorophore alone—also illustrates that increased polymeraseconcentration during polymerase-template formation results in increasedextension during the extension reaction when the complex is formed athigh temperature and in the presence of low levels of nucleotides.

In FIG. 12D, the amount of template extension obtained from twoindependent experiments, one being gel based assay and the other beingplate reader assay (FRET assay), is compared. The figure shows that theslope of the line is close to 1, thus evidencing that there is goodcorrelation between % template extension as measured by gel-based andplate-reader based assays.

Example 8: Effect of Binding Conditions on Polymerase-TemplateDissociation

This example demonstrates the effect of Sr⁺² and/or nucleotides ondissociation of the polymerase-template complex using a FRET assay (asdescribed in Example 3).

6× concentration of Pol6-67X2 (SEQ ID NO:14) was pre-incubated withLong-HP-Cy5-ExoR template (SEQ ID NOS 17 and 22). Binding was allowed toproceed for 30 minutes at 40 C in the presence of either (13A(i)) 1.2 uMdNpCpp, 3 mM SrCl2 or (13A(ii)) 1.2 uM dNpCpp or (13A(iii)) 1.2 uMpolyphosphates or (13A(iv)) absence of SrCl2, nucleotides. At time=0minutes, salt was added to a final concentration of 300 mM KGlu, andchase to a final concentration of 20×. Dissociation of template from thetemplate-DNA complex was determined following addition of polyphosphatesand MgCl2 at various time intervals.

As shown in FIGS. 13A and 13B, Sr⁺² has minimal effect on thedissociation of the polymerase from the template. Further, the lowconcentration of polyphosphate nucleotides is the best bindingcondition. Other data (not shown) illustrate that Sr⁺² does not have anysignificant effect on polymerase-template binding.

Example 8: Effect of Nucleotides and Salt Spike

This example demonstrates the effect of high nucleotide concentration onpolymerase-template binding in the presence of elevated saltconcentration.

6× concentration of Pol6-67X2 (SEQ ID NO:14) was pre-incubated withLong-HP-Cy5-ExoR template (SEQ ID NOS 17 and 22). Binding was allowed toproceed for 30 minutes at 40 C in the presence or absence of 36 uMpolyphosphates. At time=0 minutes, salt was added to a finalconcentration of either 75 mM (FIG. 14A) or 380 mM KGlu (FIG. 14B) alongwith 2 mM Biotin, 20× Chase Template, and 1 mM SrCl2. Dissociation oftemplate from he template-DNA complex was determined following additionof only Mg2+ or 36 uM polyphosphates and Mg2+ respectively at varioustime intervals.

As shown in FIG. 14A and FIG. 14B, both salt concentrations of 75KGluand 380KGlu, in the presence of high levels of nucleotides (36 μM duringbinding) resulted in 33% reduction in initial template binding. ForPol6-67X2 there does not seem to be a significant difference intemplate-polymerase dissociation rate in presence or absence ofpolyphosphates.

Example 10: Attachment of Polymerase to Nanopore

This example provides methods of attaching a variant polymerase to ananopore, e.g., α-hemolysin, OmpG.

The Pol6 variant with SpyCatcher HisTag (SEQ ID NO:4) was expressedaccording to Example 2 and purified using a cobalt affinity column. Thepolymerase-template complex was formed, purified, and attached to ananopore to form nanopore sequencing complex. Methods for formingnanopore sequencing complexes and for purifying nanopore sequencingcomplexes are described in US Provisional Application “NanoporeSequencing Complexes” 62/281,719 filed on Jan. 21, 2016, and USProvisional application “Purification of Polymerase Complexes”62/260,194 filed on Nov. 25, 2015, which are herein incorporated byreference in their entirety. Nanopore sequencing complexes can be formedby sequential binding of variant polymerase to nanopore to form anenzyme-nanopore complex, followed by association of template to form thenanopore sequencing complex. Alternatively, nanopore sequencingcomplexes can be formed by first associating the template with thevariant polymerase to form a template-enzyme complex, and subsequentlyattaching the template-enzyme complex to the nanopore.

A polymerase can be coupled to the nanopore by any suitable means. See,for example, PCT/US2013/068967 (published as WO2014/074727; GeniaTechnologies, Inc.), PCT/US2005/009702 (published as WO2006/028508;President and Fellows of Harvard College), and PCT/US2011/065640(published as WO02012/083249; Columbia University).

A variant pol6 DNA polymerase is coupled to a protein nanopore (e.g.alpha-hemolysin, OmpG), through a linker molecule. Specifically, theSpyTag and SpyCatcher system that spontaneously forms covalentisopeptide linkages under physiological conditions is used. See, forexample, Li et al, J Mol Biol. 2014 Jan. 23; 426(2):309-17.

Example 11: Nanopore Sequencing

The ability of a nanopore-bound variant Pol6 polymerase to bind taggednucleotides and thereby allow for the detection of blocked channelcurrents at the nanopore to which the polymerase is attached, wasdetermined. Increased processivity of the variant Pol6 polymerases wascompared to that of the parent P016 lacking the modifications of thevariant enzyme.

The variant Pol6 polymerase is contacted with DNA template to formvariant Pol6-DNA complex, which is subsequently attached to a nanoporeembedded in a lipid bilayer over a well on a semiconductor sensor chip,also called a biochip. The lipid bilayer is formed and the nanopore withattached variant Pol6 polymerase-DNA complex i.e. the variant Pol6nanopore sequencing complex, is inserted as described inPCT/US2014/061853 (entitled “Methods for Forming Lipid Bilayers onBiochips” and filed 22 Oct. 2014).

Alternatively, the nanopore is embedded into the lipid bilayer, and thevariant Pol6-DNA complex is attached in situ.

A mixture of tagged nucleotides, where the tag is a polymer of 30thymine nucleotides (T30) consisting of 3 uM T-T30, 3 uM C-T30, 3 uMG-T30, and 3 uM A-T30, in static conditions (500 mM KGlu, 3 mM CaCl₂, 20mM HEPES, pH8.0), is flowed over the nanopores at a rate of 0.834ul/second.

An alternating current of 210 mV peak to peak is applied at 25 Hz, andcapture of nucleotide tags is assessed as nucleotide bases areincorporated into the copied DNA strand by the nanopore-boundpolymerase.

Processivity of the variant Pol6 is compared to that of the unmodifiedparental Pol6 to determine an increase in read-length, and/or speed ofpolynucleotide synthesis, and/or a decrease in sequencing error.

SEQUENCE LISTING FREE TEXTSEQ ID NO: 1-Wild-type Pol6 (DNA polymerase [Clostridium phage phiCPV4];GenBank: AFH27113.1)001 mdkhtqyvke hsfnydeykk anfdkiecli fdtesctnye ndntgarvyg wglgvtrnhn061 miygqnlnqf wevcqnifnd wyhdnkhtik itktkkgfpk rkyikfpiav hnlgwdvefl121 hyslvengfn ydkgllktvf skgapyqtvt devvpktfhi vqnnnivygc nvymdkffev181 enkdgsttei glcldffdsy kiitcaesqf hnyvhdvdpm fymkgeeydy dtwrspthkq241 ttlelryqyn diymlrevie qfyidglcgg elpltgmrta ssiagnvlkk mtfgeektee301 gyinyfeldk ktkfeflrkr iemesytggy thanhkavgk tinkigcsld inssypsqma361 ykvfpygkpv rktwgrkpkt eknevyliev gfdfvepkhe eyaldifkig avnskalspi421 tgavsgqeyf ctnikdgkai pvykelkdtk lttnynvvlt sveyefwikh fnfgvfkkde481 ydcfevdnle ftglkigsil yykaekgkfk pyvdhtfkmk venkklgnkp ltnqakliln541 gaygkfgtkq nkeekdlimd knglltftgs vteyegkefy fpyasfvtay grlqlwnaii601 yavgvenfly cdtdsiycnr evnsliedmn aigetidkti lgkwdvehvf dkfkvlgqkk661 ymyhdckedk tdlkccglps darkiiigqg fdefylgknv egkkqrkkvi ggcllldtlf721 tikkimf* SEQ ID NO: 2-Pol6 (with His tag)MHHHHHHHHS GGSDKHTQYV KEHSFNYDEY KKANFDKIEC LIFDTESCTN  50YENDNTGARV YGWGLGVTRN HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT 100IKITKTKKGF PKRKYIKFPI AVHNLGWDVE FLKYSLVENG FNYDKGLLKT 150VFSKGAPYQT VTDVEEPKTF HIVQNNNIVY GCNVYMDKFF EVENKDGSTT 200EIGLCLDFFD SYKIITCAES QFHNYVHDVD PMFYKMGEEY DYDTWRSPTH 250KQTTLELRYQ YNDIYMLREV IEQFYIDGLC GGELPLTGMR TASSIAFNVL 300KKMTFGEEKT EEGYINYFEL DKKTKFEFLR KRIEMESYTG GYTHANHKAV 350GKTINKIGCS LDINSSYPSQ MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI 400EVGFDFVEPK HEEYALDIFK IGAVNSKALS PITGAVSGQE YFCTNIKDGK 450AIPVYKELKD TKLTTNYNVV LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN 500LEFTGLKIGS ILYYKAEKGK FKPYVDHFTK MKVENKKLGN KPLTNQAKLI 550LNGAYGKFGT KQNKEEKDLI MDKNGLLTFT GSVTEYEGKE FYRPYASFVT 600AYGRLQLWNA IIYAVGVENF LYCDTDSIYC NREVNSLIED MNAIGETIDK 650TILGKWDVEH VFDKFKVLGQ KKYMYHDCKE DKTDLKCCGL PSDARKIIIG 700QGFDEFYLGK NVEGKKQRKK VIGGCLLLDT LFTIKKIMF*            739SEQ ID NO: 3-Pol6 with His-tag (DNA sequence) ATGCATCACC ATCATCATCA CCACCAC AGC GGCGGTTCCG ACAAACACAC   50GCAGTACGTC AAAGAGCATA GCTTCAATTA TGACGAGTAT AAGAAAGCGA  100ATTTCGACAA GATCGAGTGC CTGATCTTTG ACACCGAGAG CTGCACGAAT  150TATGAGAACG ATAATACCGG TGCACGTGTT TACGGTTGGG GTCTTGGCGT  200CACCCGCAAC CACAATATGA TCTACGGCCA AAATCTGAAT CAGTTTTGGG  250AAGTATGCCA GAACATTTTC AATGATTGGT ATCACGACAA CAAACATACC  300ATTAAGATTA CCAAGACCAA GAAAGGCTTC CCGAAACGTA AGTACATTAA  350GTTTCCGATT GCAGTTCACA ATTTGGGCTG GGATGTTGAA TTCCTGAAGT  400ATAGCCTGGT GGAGAATGGT TTCAATTACG ACAAGGGTCT GCTGAAAACT  450GTTTTTAGCA AGGGTGCGCC GTACCAAACC GTGACCGATG TTGAGGAACC  500GAAAACGTTC CATATCGTCC AGAATAACAA CATCGTTTAT GGTTGTAACG  550TGTATATGGA CAAATTCTTT GAGGTCGAGA ACAAAGACGG CTCTACCACC  600GAGATTGGCC TGTGCTTGGA TTTCTTCGAT AGCTATAAGA TCATCACGTG  650TGCTGAGAGC CAGTTCCACA ATTACGTTCA TGATGTGGAT CCAATGTTCT  700ACAAAATGGG TGAAGAGTAT GATTACGATA CTTGGCGTAG CCCGACGCAC  750AAGCAGACCA CCCTGGAGCT GCGCTACCAA TACAATGATA TCTATATGCT  800GCGTGAAGTC ATCGAACAGT TTTACATTGA CGGTTTATGT GGCGGCGAGC  850TGCCGCTGAC CGGCATGCGC ACCGCTTCCA GCATTGCGTT CAACGTGCTG  900AAAAAGATGA CCTTTGGTGA GGAAAAGACG GAAGAGGGCT ACATCAACTA  950TTTTGAATTG GACAAGAAAA CCAAATTCGA GTTTCTGCGT AAGCGCATTG 1000AAATGGAATC GTACACCGGT GGCTATACGC ACGCAAATCA CAAAGCCGTT 1050GGTAAGACTA TTAACAAGAT CGGTTGCTCT TTGGACATTA ACAGCTCATA 1100CCCTTCGCAG ATGGCGTACA AGGTCTTTCC GTATGGCAAA CCGGTTCGTA 1150AGACCTGGGG TCGTAAACCA AAGACCGAGA AGAACGAAGT TTATCTGATT 1200GAAGTTGGCT TTGACTTCGT GGAGCCGAAA CACGAAGAAT ACGCGCTGGA 1250TATCTTTAAG ATTGGTGCGG TGAACTCTAA AGCGCTGAGC CCGATCACCG 1300GCGCTGTCAG CGGTCAAGAG TATTTCTGTA CGAACATTAA AGACGGCAAA 1350GCAATCCCGG TTTACAAAGA ACTGAAGGAC ACCAAATTGA CCACTAACTA 1400CAATGTCGTG CTGACCAGCG TGGAGTACGA GTTCTGGATC AAACACTTCA 1450ATTTTGGTGT GTTTAAGAAA GACGAGTACG ACTGTTTCGA AGTTGACAAT 1500CTGGAGTTTA CGGGTCTGAA GATTGGTTCC ATTCTGTACT ACAAGGCAGA 1550GAAAGGCAAG TTTAAACCTT ACGTGGATCA CTTCACGAAA ATGAAAGTGG 1600AGAACAAGAA ACTGGGTAAT AAGCCGCTGA CGAATCAGGC AAAGCTGATT 1650CTGAACGGTG CGTACGGCAA ATTCGGCACC AAACAAAACA AAGAAGAGAA 1700AGATTTGATC ATGGATAAGA ACGGTTTGCT GACCTTCACG GGTAGCGTCA 1750CGGAATACGA GGGTAAAGAA TTCTATCGTC CGTATGCGAG CTTCGTTACT 1800GCCTATGGTC GCCTGCAACT GTGGAACGCG ATTATCTACG CGGTTGGTGT 1850GGAGAATTTT CTGTACTGCG ACACCGACAG CATCTATTGT AACCGTGAAG 1900TTAACAGCCT CATTGAGGAT ATGAACGCCA TTGGTGAAAC CATCGATAAA 1950ACGATTCTGG GTAAATGGGA CGTGGAGCAT GTCTTTGATA AGTTTAAGGT 2000CCTGGGCCAG AAGAAGTACA TGTATCATGA TTGCAAAGAA GATAAAACGG 2050ACCTGAAGTG TTGCGGTCTG CCGAGCGATG CCCGTAAGAT TATCATTGGT 2100CAAGGTTTCG ACGAGTTTTA TCTGGGCAAA AATGTCGAAG GTAAGAAGCA 2150ACGCAAAAAA GTGATCGGCG GTTGCCTGCT GCTGGACACC CTGTTTACGA 2200TCAAGAAAAT CATGTTCTAA                                  2220SEQ ID NO: 4-Pol6-44-X1 with His-tag/SpyCatcher MHHHHHHHH

   50

   100

 GGSDKHTQYV  150KEHSFNYDEY KKANFDKIEC LIFATESCTN YENDNTGARV YGWGLGVTRN  200HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT IKITKTKKGF PKRKYIKFPI  250AVHNLGWDVE FLKYSLVENG FNYDKGLLKT VFSKGAPYQT VTDVEEPKTF  300HIVQNNNIVY GCNVYMDKFF EVENKDGSTT EIGLCLDFFD EYKIITCAES  350QFHNYVHDVD PMFYKMGEEY DYDTWRSPTH KQTTLELRYQ YNDIYMLREV  400IEQFYIDGLC GGELPLTGMR TASSIAFNVL KKMTFGEEKT EEGYINYFEL  450DKKTKFEFLR KRIEMESYTG GYTHANHKAV GKTINKIGCS LDINSAYPSQ  500MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI EVGFDFVEPK HEEYALDIFK  550IGAVNSKALS PITGAVSGQE IFCTNIKDGK AIPVYKELKD TKLTTNYNVV  600LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN LEFTGLKIGS ILYYKAEKGK  650FKPYVDHFMK MKVENKKLGN KPLTNQFKLI LNGAYGKFGT KQNKEEKDLI  700MDKNGLLTFT GSVT

YEGKE FYRPYASFVT AYGRLQLWNA IIYAVGVENF  750LYCDTDSIYC NREVNSLIED MNAIGETIDK TILGKWDVEH VFDKFKVLGQ  800KKYMYHDCKE DKTDLKCCGL PSDARKIIIG QGFDEFYLGK NVEGKKQRKK  850 VIGGCLLLDT 

FTIKKI

F*                                   869SEQ ID NO: 5-Pol6-44-X1 with His-tag/SpyCatcher (DNA sequence)ATGCATCACC ATCATCATCA CCACCAC

   50

    100

    150

    200

    250

     300

     350

     400

 GGCGGTTCCG ACAAACACAC GCAGTACGTC  450AAAGAGCATA GCTTCAATTA TGACGAGTAT AAGAAAGCGA ATTTCGACAA  500GATCGAGTGC CTGATCTTTG CGACCGAGAG CTGCACGAAT TATGAGAACG  550ATAATACCGG TGCACGTGTT TACGGTTGGG GTCTTGGCGT CACCCGCAAC  600CACAATATGA TCTACGGCCA AAATCTGAAT CAGTTTTGGG AAGTATGCCA  650GAACATTTTC AATGATTGGT ATCACGACAA CAAACATACC ATTAAGATTA  700CCAAGACCAA GAAAGGCTTC CCGAAACGTA AGTACATTAA GTTTCCGATT  750GCAGTTCACA ATTTGGGCTG GGATGTTGAA TTCCTGAAGT ATAGCCTGGT  800GGAGAATGGT TTCAATTACG ACAAGGGTCT GCTGAAAACT GTTTTTAGCA  850AGGGTGCGCC GTACCAAACC GTGACCGATG TTGAGGAACC GAAAACGTTC  900CATATCGTCC AGAATAACAA CATCGTTTAT GGTTGTAACG TGTATATGGA  950CAAATTCTTT GAGGTCGAGA ACAAAGACGG CTCTACCACC GAGATTGGCC 1000TGTGCTTGGA TTTCTTCGAT AGCTATAAGA TCATCACGTG TGCTGAGAGC 1050CAGTTCCACA ATTACGTTCA TGATGTGGAT CCAATGTTCT ACAAAATGGG 1100TGAAGAGTAT GATTACGATA CTTGGCGTAG CCCGACGCAC AAGCAGACCA 1150CCCTGGAGCT GCGCTACCAA TACAATGATA TCTATATGCT GCGTGAAGTC 1200ATCGAACAGT TTTACATTGA CGGTTTATGT GGCGGCGAGC TGCCGCTGAC 1250CGGCATGCGC ACCGCTTCCA GCATTGCGTT CAACGTGCTG AAAAAGATGA 1300CCTTTGGTGA GGAAAAGACG GAAGAGGGCT ACATCAACTA TTTTGAATTG 1350GACAAGAAAA CCAAATTCGA GTTTCTGCGT AAGCGCATTG AAATGGAATC 1400GTACACCGGT GGCTATACGC ACGCAAATCA CAAAGCCGTT GGTAAGACTA 1450TTAACAAGAT CGGTTGCTCT TTGGACATTA ACAGCGCGTA CCCTTCGCAG 1500ATGGCGTACA AGGTCTTTCC GTATGGCAAA CCGGTTCGTA AGACCTGGGG 1550TCGTAAACCA AAGACCGAGA AGAACGAAGT TTATCTGATT GAAGTTGGCT 1600TTGACTTCGT GGAGCCGAAA CACGAAGAAT ACGCGCTGGA TATCTTTAAG 1650ATTGGTGCGG TGAACTCTAA AGCGCTGAGC CCGATCACCG GCGCTGTCAG 1700CGGTCAAGAG TATTTCTGTA CGAACATTAA AGACGGCAAA GCAATCCCGG 1750TTTACAAAGA ACTGAAGGAC ACCAAATTGA CCACTAACTA CAATGTCGTG 1800CTGACCAGCG TGGAGTACGA GTTCTGGATC AAACACTTCA ATTTTGGTGT 1850GTTTAAGAAA GACGAGTACG ACTGTTTCGA AGTTGACAAT CTGGAGTTTA 1900CGGGTCTGAA GATTGGTTCC ATTCTGTACT ACAAGGCAGA GAAAGGCAAG 1950TTTAAACCTT ACGTGGATCA CTTCATGAAA ATGAAAGTGG AGAACAAGAA 2000ACTGGGTAAT AAGCCGCTGA CGAATCAGTT TAAGCTGATT CTGAACGGTG 2050CGTACGGCAA ATTCGGCACC AAACAAAACA AAGAAGAGAA AGATTTGATC 2100ATGGATAAGA ACGGTTTGCT GACCTTCACG GGTAGCGTCA CGGAATACGA 2150GGGTAAAGAA TTCTATCGTC CGTATGCGAG CTTCGTTACT GCCTATGGTC 2200GCCTGCAACT GTGGAACGCG ATTATCTACG CGGTTGGTGT GGAGAATTTT 2250CTGTACTGCG ACACCGACAG CATCTATTGT AACCGTGAAG TTAACAGCCT 2300CATTGAGGAT ATGAACGCCA TTGGTGAAAC CATCGATAAA ACGATTCTGG 2350GTAAATGGGA CGTGGAGCAT GTCTTTGATA AGTTTAAGGT CCTGGGCCAG 2400AAGAAGTACA TGTATCATGA TTGCAAAGAA GATAAAACGG ACCTGAAGTG 2450TTGCGGTCTG CCGAGCGATG CCCGTAAGAT TATCATTGGT CAAGGTTTCG 2500ACGAGTTTTA TCTGGGCAAA AATGTCGAAG GTAAGAAGCA ACGCAAAAAA 2550GTGATCGGCG GTTGCCTGCT GCTGGACACC CTGTTTACGA TCAAGAAAAT 2600CATGTTCTAA                                             2610SEQ ID NO: 6-Pol6-44-X1 with His-tag/SpyCatcher + E585K of SEQ IDNO: 2, which corresponds to E715K of SEQ ID NO: 6 MHHHHHHHH

    50

    100

 GGSDKHTQYV   150KEHSFNYDEY KKANFDKIEC LIFATESCTN YENDNTGARV YGWGLGVTRN  200HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT IKITKTKKGF PKRKYIKFPI  250AVHNLGWDVE FLKYSLVENG FNYDKGLLKT VFSKGAPYQT VTDVEEPKTF  300HIVQNNNIVY GCNVYMDKFF EVENKDGSTT EIGLCLDFFD SYKIITCAES  350QFHNYVHDVD PMFYKMGEEY DYDTWRSPTH KQTTLELRYQ YNDIYMLREV  400IEQFYIDGLC GGELPLTGMR TASSIAFNVL KKMTFGEEKT EEGYINYFEL  450DKKTKFEFLR KRIEMESYTG GYTHANHKAV GKTINKIGCS LDINSAYPSQ  500MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI EVGFDFVEPK HEEYALDIFK  550IGAVNSKALS PITGAVSGQE YFCTNIKDGK AIPVYKELKD TKLTTNYNVV  600LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN LEFTGLKIGS ILYYKAEKGK  650FKPYVDHFMK MKVENKKLGN KPLTNQFKLI LNGAYGKFGT KQNKEEKDLI  700MDKNGLLTFT GSVT

YEGKE FYRPYASFVT AYGRLQLWNA IIYAVGVENF  750LYCDTDSIYC NREVNSLIED MNAIGETIDK TILGKWDVEH VFDKFKVLGQ  800KKYMYHDCKE DKTDLKCCGL PSDARKIIIG QGFDEFYLGK NVEGKKQRKK  850VIGGCLLLDT LFTIKKIMF*                                   869SEQ ID NO: 7-Pol6-44-X1 with His-tag/SpyCatcher + E585K + L731K ofSEQ ID NO: 2, which correspond to E715K + L861K of SEQ ID NO: 6MHHHHHHHHS GDYDIPTTEN LYFQGAMVDT LSGLSSEQGQ SGDMTIEEDS   50ATHIKFSKRD EDGKELAGAT MELRDSSGKT ISTWISDGQV KDFYLYPGKY  100TFVETAAPDG YEVATAITFT VNEQGQVTVN GKATKGDAHI GGSDKHTQYV  150KEHSFNYDEY KKANFDKIEC LIFATESCTN YENDNTGARV YGWGLGVTRN  200HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT IKITKTKKGF PKRKYIKFPI  250AVHNLGWDVE FLKYSLVENG FNYDKGLLKT VFSKGAPYQT VTDVEEPKTF  300HIVQNNNIVY GCNVYMDKFF EVENKDGSTT EIGLCLDFFD SYKIITCAES  350QFHNYVHDVD PMFYKMGEEY DYDTWRSPTH KQTTLELRYQ YNDIYMLREV  400IEQFYIDGLC GGELPLTGMR TASSIAFNVL KKMTFGEEKT EEGYINYFEL  450DKKTKFEFLR KRIEMESYTG GYTHANHKAV GKTINKIGCS LDINSAYPSQ  500MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI EVGFDFVEPK HEEYALDIFK  550IGAVNSKALS PITGAVSGQE YFCTNIKDGK APIVYKELKD TKLTTNYNVV  600LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN LEFTGLKIGS ILYYKAEKGK  650FKPYVDHFMK MKVENKKLGN KPLTNQFKLI LNGAYGKFGT KQNKEEKDLI  700MDKNGLLTFT GSVT

YEGKE FYRPYASFVT AYGRLQLWNA IIYAVGVENF  750LYCDTDSIYC NREVNSLIED MNAIGETIDK TILGKWDVEH VFDKFKVLGQ  800KKYMYHDCKE DKTDLKCCGL PSDARKIIIG QGFDEFYLGK NVEGKKQRKK  850 VIGGCLLLDT 

FTIKKIMF*                                   869SEQ ID NO: 8-Pol6-44-X1 with His-tag/SpyCatcher + E585K + M738K ofSEQ ID NO: 2, which correspond to E715K + M868K of SEQ ID NO: 6MHHHHHHHH

    50

    100

 GGSDKHTQYV   150KEHSFNYDEY KKANFDKIEC LIFATESCTN YENDNTGARV YGWGLGVTRN  200HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT IKITKTKKGF PKRKYIKFPI  250AVHNLGWDVE FLKYSLVENG FNYDKGLLKT VFSKGAPYQT VTDVEEPKTF  300HIVQNNNIVY GCNVYMDKFF EVENKDGSTT EIGLCLDFFD SYKIITCAES  350QFHNYVHDVD PMFYKMGEEY DYDTWRSPTH KQTTLELRYQ YNDIYMLREV  400IEQFYIDGLC GGELPLTGMR TASSIAFNVL KKMTFGEEKT EEGYINYFEL  450DKKTKFEFLR KRIEMESYTG GYTHANHKAV GKTINKIGCS LDINSAYPSQ  500MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI EVGFDFVEPK HEEYALDIFK  550IGAVNSKALS PITGAVSGQE YFCTNIKDGK AIPVYKELKD TKLTTNYNVV  600LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN LEFTGLKIGS ILYYKAEKGK  650FKPYVDHFMK MKVENKKLGN KPLTNQFKLI LNGAYGKFGT KQNKEEKDLI  700MDKNGLLTFT GSVT

YEGKE FYRPYASFVT AYGRLQLWNA IIYAVGVENF  750LYCDTDSIYC NREVNSLIED MNAIGETIDK TILGKWDVEH VFDKFKVLGQ  800KKYMYHDCKE DKTDLKCCGL PSDARKIIIG QGFDEFYLGK NVEGKKQRKK  850VIGGCLLLDT LFTIKKI

F*                                   869 SEQ ID NO: 9-His 6 tag: HHHHHHSEQ ID NO: 10-SpyCatcherSGDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHISEQ ID NO: 11-SpyTag: AHIVMVDAYKPTKSEQ ID NOS 12 and 21-Cy5-labelled fluorogenic DNA templateCy5/AGA GTG ATA GTA TGA TTA TGT AGA TGT AGG ATT TGA TAT GTG AGT AGCCGA ATG AAA CCT T/iSpC3/TT GGT TTC ATT CGGSEQ ID NO: 13-Black Hole Quencher ® dye-labelled quencheroligonucleotide TTT TCA TAA TCA TAC TAT CAC TCT/3BHQ_2SEQ ID NO: 14-Pol6-67 X2 (with His tag and T529M-S366A-A547F-N545L-Y225L-D657R Y242A)MHHHHHHHHS GGSDKHTQYV KEHSFNYDEY KKANFDKIEC LIFDTESCTN  50YENDNTGARV YGWGLGVTRN HNMIYGQNLN QFWEVCQNIF NDWYHDNKHT 100IKITKTKKGF PKRKYIKFPI AVHNLGWDVE FLKYSLVENG FNYDKGLLKT 150VFSKGAPYQT VTDVEEPKTF HIVQNNNIVY GCNVYMDKFF EVENKDGSTT 200EIGLCLDFFD SYKIITCAES QFHNLVHDVD PMFYKMGEEY DADTWRSPTH 250KQTTLELRYQ YNDIYMLREV IEQFYIDGLC GGELPLTGMR TASSIAFNVL 300KKMTFGEEKT EEGYINYFEL DKKTKFEFLR KRIEMESYTG GYTHANHKAV 350GKTINKIGCS LDINSAYPSQ MAYKVFPYGK PVRKTWGRKP KTEKNEVYLI 400EVGFDFVEPK HEEYALDIFK IGAVNSKALS PITGAVSGQE YFCTNIKDGK 450AIPVYKELKD TKLTTNYNVV LTSVEYEFWI KHFNFGVFKK DEYDCFEVDN 500LEFTGLKIGS ILYYKAEKGK FKPYVDHFMK MKVENKKLGN KPLTLQFKLI 550LNGAYGKFGT KQNKEEKDLI MDKNGLLTFT GSVTEYEGKE FYRPYASFVT 600AYGRLQLWNA IIYAVGVENF LYCDTDSIYC NREVNSLIED MNAIGETIDK 650TILGKWRVEH VFDKFKVLGQ KKYMYHDCKE DKTDLKCCGL PSDARKIIIG 700QGFDEFYLGK NVEGKKQRKK VIGGCLLLDT LFTIKKIMF*            739SEQ ID NOS 15 and 22-Fluorescent Hairpin DNA template/5deSBioTEG/ACTGCTGATCTGTTCCTGAATCGACTACTACTATCATCATACCACCTCAGCTGCACG/iFluorT/T/iSpC3/AAGTGCAGCTGAGGTGG SEQ ID NO: 16-Chase TemplateAGAGTGATAGTATGATTATGTATGTGAGTAGTCCACTGAAACCTTTGGTTTCAGTGGA/3ddC/SEQ ID NOS 17 and 22-LongHP-Cy5-ExoR/5Cy5/ATCTCTTCAACTCGACTTATGTTCTACTGCTGATCTGTTCCTGAATCGACTACTACTATCATCATACCACCTCAGCTGCACGT/iSpC3/AAGTGCAGCTGAGGTGGSEQ ID NO: 18-Quencher PrimerTTTGATTCAGGAACAGATCAGCAGTAGAACATAAGTCGAGTTGAAGAGAT/3BHQ_2/

What is claimed is:
 1. A method for preparing a polymerase-templatecomplex, comprising: (a) providing a polymerase; and (b) contacting thepolymerase with a polynucleotide template in a solution comprising a lowconcentration of nucleotides and a high temperature, thereby preparingthe polymerase-template complex.
 2. The method of claim 1, furthercomprising saturating the solution with the polymerase of thepolymerase-template complex.
 3. The method of claim 1, wherein theconcentration of nucleotides is 0.8 μM to 2.2 μM.
 4. The method of claim1, wherein the high temperature of the solution is a temperature aboveroom temperature.
 5. The method of claim 4, wherein the temperature ofthe solution is 35° C. to 45° C.
 6. The method of claim 1, furthercomprising raising the concentration of nucleotides in the solutionafter preparing the polymerase-template complex.
 7. The method of claim1, wherein the polymerase of the polymerase-template complex has atleast 85%, 90%, 95%, 98% or more sequence identity to the amino acidsequence set forth as SEQ ID NO:
 14. 8. A method for increasingprocessivity of a template-polymerase complex, the method comprisingforming a polymerase-template complex in a solution comprising a lowconcentration of nucleotides and a high temperature, wherein theprocessivity of the polymerase-template complex formed in thehigh-temperature solution is greater than a processivity resulting froma control polymerase-template complex solution at room temperature. 9.The method of 8, wherein the concentration of nucleotides is about 1.2μM.
 10. The method of claim 8, wherein the temperature of the solutionis about 40° C.
 11. The method of claim 8, further comprising raisingthe concentration of nucleotides in the solution after forming thepolymerase-template complex.
 12. The method of claim 8, wherein thepolymerase of the polymerase-template complex has at least 85%, 90%,95%, 98% or more sequence identity to the amino acid sequence set forthas SEQ ID NO:
 14. 13. A method for nanopore-based sequencing of apolynucleotide template, the method comprising: forming apolymerase-template complex in a solution comprising a low concentrationof nucleotides, the solution having a high temperature; combining theformed polymerase-template complex with a nanopore to form ananopore-sequencing complex; providing tagged nucleotides to thenanopore sequencing complex to initiate template-dependent nanoporesequencing of the template at the high temperature; detecting, with theaid of the nanopore, a tag associated with each of the taggednucleotides during incorporation of each of the tagged nucleotides whileeach of the tagged nucleotides is associated with the polymerase,thereby determining the sequence of the polynucleotide template.
 14. Themethod of claim 13, wherein forming the polymerase-template complexcomprises saturating the solution with the polymerase of thepolymerase-template complex.
 15. The method of claim 13, wherein theconcentration of nucleotides is 0.8 μM to 2.2 μM.
 16. The method of 15,wherein the concentration of nucleotides is 1.2 μM.
 17. The method ofclaim 13, wherein the temperature of the solution is about 40° C. 18.The method of claim 13, wherein the polymerase of thepolymerase-template complex has at least 85%, 90%, 95%, 98% or moresequence identity to the amino acid sequence set forth as SEQ ID NO: 14.19. The method of claim 13, wherein the nanopore is an oligomericnanopore.
 20. The method of claim 19, wherein the nanopore is analpha-hemolysin nanopore.